Chimeric Receptors with 4-1BB Stimulatory Signaling Domain

ABSTRACT

The present invention relates to a chimeric receptor capable of signaling both a primary and a co-stimulatory pathway, thus allowing activation of the co-stimulatory pathway without binding to the natural ligand. The cytoplasmic domain of the receptor contains a portion of the 4-1BB signaling domain. Embodiments of the invention relate to polynucleotides that encode the receptor, vectors and host cells encoding a chimeric receptor, particularly including T cells and natural killer (NK) cells and methods of use.

2. CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/301,122, filed Jun. 10, 2014, which is a continuation of U.S.application Ser. No. 13/761,917, filed Feb. 7, 2013, which is adivisional application of U.S. application Ser. No. 13/548,148, filedJul. 12, 2012 (granted as U.S. Pat. No. 8,399,645), which is acontinuation of U.S. application Ser. No. 13/244,981, filed Sep. 26,2011 (abandoned), which is a continuation of U.S. patent applicationSer. No. 12/206,204, filed on Sep. 8, 2008 (granted as U.S. Pat. No.8,026,097), which is a continuation of U.S. Patent Application No.11/074,525, filed on Mar. 8, 2005 (granted as U.S. Pat. No. 7,435,596),which is a continuation-in-part of U.S. patent application Ser. No.10/981,352 filed Nov. 4, 2004 (abandoned), each of which is incorporatedherein by reference in its entirety.

1. GOVERNMENT INTEREST

This invention was made in part with U.S. Government support underNational Institutes of Health grant No. CA 58297. The U.S. Governmentmay have certain rights in this invention.

3. FIELD OF THE INVENTION

This invention relates to chimeric cell membrane receptors, particularlychimeric T-cell receptors. This invention further relates to activationand expansion of cells for therapeutic uses, in particular foractivation and expansion of NK cells for chimeric receptor-based celltherapy.

4. BACKGROUND

Regulation of cell activities is frequently achieved by the binding of aligand to a surface membrane receptor comprising an extracellular and acytoplasmic domain. The formation of the complex between the ligand andthe extracellular portion of the receptor results in a conformationalchange in the cytoplasmic portion of the receptor which results in asignal transduced within the cell. In some instances, the change in thecytoplasmic portion results in binding to other proteins, where otherproteins are activated and may carry out various functions. In somesituations, the cytoplasmic portion is autophosphorylated orphosphorylated, resulting in a change in its activity. These events arefrequently coupled with secondary messengers, such as calcium, cyclicadenosine monophosphate, inositol phosphate, diacylglycerol, and thelike. The binding of the ligand to the surface membrane receptor resultsin a particular signal being transduced.

For T-cells, engagement of the T-cell receptor (TCR) alone is notsufficient to induce persistent activation of resting naive or memory Tcells. Full, productive T cell activation requires a secondco-stimulatory signal from a competent antigen-presenting cell (APC).Co-stimulation is achieved naturally by the interaction of theco-stimulatory cell surface receptor on the T cell with the appropriatecounter-receptor on the surface of the APC. An APC is normally a cell ofhost origin which displays a moiety which will cause the stimulation ofan immune response. APCs include monocyte/macrophages, dendritic cells,B cells, and any number of virally-infected or tumor cells which expressa protein on their surface recognized by T cells. To be immunogenic APCsmust also express on their surface a co-stimulatory molecule. Such APCsare capable of stimulating T cell proliferation, inducing cytokineproduction, and acting as targets for cytolytic T cells upon directinteraction with the T cell. See Linsley and Ledbetter, Ann. Rev.Immunol. 4:191-212 (1993); Johnson and Jenkins, Life Sciences55:1767-1780 (1994); June et al., Immunol. Today 15:321-331 (1994); andMondino and Jenkins, J. Leuk. Biol. 55:805-815 (1994).

Engagement of the co-stimulatory molecule together with the TCR isnecessary for optimal levels of IL-2 production, proliferation andclonal expansion, and generation of effector functions such as theproduction of immunoregulatory cytokines, induction of antibodyresponses from B cells, and induction of cytolytic activity. Moreimportantly, engagement of the TCR in the absence of the co-stimulatorysignal results in a state of non-responsiveness, called anergy. Anergiccells fail to become activated upon subsequent stimulation through theTCR, even in the presence of co-stimulation, and in some cases may beinduced to die by a programmed self-destruct mechanism.

In certain situations, for example where APCs lack the counter-receptormolecules necessary for co-stimulation, it would be beneficial to havethe co-stimulatory signal induced by virtue of employing a ligand otherthan the natural ligand for the co-stimulatory receptor. This might be,for example, the same ligand as that recognized by the TCR (i.e., thesame moiety, such that if one signal is received, both signals will bereceived), or another cell surface molecule known to be present on thetarget cells (APCs).

Several receptors that have been reported to provide co-stimulation forT-cell activation, including CD28, OX40, CD27, CD2, CD5, ICAM-1, LFA-1(CD11a/CD18), and 4-1BB. The signaling pathways utilized by theseco-stimulatory molecules share the common property of acting in synergywith the primary T cell receptor activation signal.

Previously the signaling domain of CD28 has been combined with theT-cell receptor to form a co-stimulatory chimeric receptor. See U.S.Pat. No. 5,686,281; Geiger, T. L. et al., Blood 98: 2364-2371 (2001);Hombach, A. et al., J Immunol 167: 6123-6131 (2001); Maher, J. et al.Nat Biotechnol 20: 70-75 (2002); Haynes, N. M. et al., J Immunol 169:5780-5786 (2002); Haynes, N. M. et al., Blood 100: 3155-3163 (2002).These co-stimulatory receptors provide a signal that is synergistic withthe primary effector activation signal, i.e. the TCR signal or thechimeric effector function receptor signal, and can complete therequirements for activation under conditions where stimulation of theTCR or chimeric effector function receptor is suboptimal and mightotherwise be detrimental to the function of the cell. These receptorscan support immune responses, particularly of T cells, by permitting theuse of ligands other than the natural ligand to provide the requiredco-stimulatory signal.

Chimeric receptors that contain a CD19 specific single chainimmunoglobulin extracellular domain have been shown to lyse CD19+ targetcells and eradicate CD19+ B cell lymphomas engrafted in mice [Cooper LJ, et al., Blood 101:1637-1644 (2003) and Brentjens R J, et al., NatureMedicine 9:279-286 (2003)]. Cooper et al. reported that T-cell clonestransduced with chimeric receptors comprising anti-CD19 scFv and CD3ζproduced approximately 80% specific lysis of B-cell leukemia andlymphoma cell lines at a 1:1 effector to target ratio in a 4-hour Crrelease assay; at this ratio, percent specific lysis of one primaryB-lineage ALL sample tested was approximately 30%. Brentjens et al.reported that T-cells bearing anti-CD19 scFv and CD3ζ chimeric receptorscould be greatly expanded in the presence of exogenous IL-15 andartificial antigen-presenting cells transduced with CD19 and CD80. Theauthors showed that these T cells significantly improved the survival ofimmunodeficient mice engrafted with the Raji B-cell lymphoma cell line.Their results also confirmed the importance of co-stimulation inmaximizing T-cell-mediated anti-leukemic activity. Only cells expressingthe B7 ligands of CD28 elicited effective T-cell responses. This couldbe a major obstacle in the case of B-lineage ALL because leukemiclymphoblasts typically do not express B7 molecules.

In addition to T cell immune responses, natural killer (NK) cellresponses appear to be clinically relevant. While T cells recognizetumor associated peptide antigen expressed on surface HLA class I orclass II molecules, antigen nonspecific immune responses are mediated byNK cells that are activated by the failure to recognize cognate “self”HLA class I molecules. The graft-versus-tumor effect of transplantsusing HLA matched donors is mediated by antigen specific T cells, whiletransplantation using HLA mismatched donors can also lead to donor NKcells with potent antitumor activity. HLA mismatched haplo-identicaltransplants can exert a powerful anti-leukemia effect based on expansionof antigen nonspecific donor NK cells.

Immunotherapy with NK cells has been limited by the inability to obtainsufficient numbers of pure NK cells suitable for manipulation andexpansion. The established methods for cell expansion favor T cellexpansion and even after T cells are depleted, residual T cellstypically become prominent after stimulation. Thus there is a need forbetter methods to expand NK cells from a population without expanding Tcells.

5. SUMMARY OF THE INVENTION

The present invention provides a chimeric receptor containing aco-stimulatory signal by incorporation of the signaling domain of the4-1BB receptor. The chimeric receptor comprises an extracellular ligandbinding domain, a transmembrane domain and a cytoplasmic domain whereinthe cytoplasmic domain comprises the signaling domain of 4-1BB. In oneembodiment of the invention the signaling domain of 4-1BB used in thechimeric receptor is of human origin. In a preferred embodiment, human4-1BB consists of SEQ ID NO:2. In another embodiment the signalingdomain comprises amino acids 214-255 of SEQ ID NO:2.

In another embodiment of the invention the cytoplasmic domain of thechimeric receptor comprises the signaling domain of CD3ζ in addition tothe signaling domain of 4-1BB. In another embodiment the extracellulardomain comprises a single chain variable domain of an anti-CD19monoclonal antibody. In another embodiment the transmembrane domaincomprises the hinge and transmembrane domains of CD8α. In a mostpreferred embodiment of the invention the extracellular domain comprisesa single chain variable domain of an anti-CD 19 monoclonal antibody, thetransmembrane domain comprises the hinge and transmembrane domain ofCD8α, and the cytoplasmic domain comprises the signaling domain of CD3ζand the signaling domain of 4-1BB.

Other aspects of the invention include polynucleotide sequences, vectorsand host cells encoding a chimeric receptor that comprises the signalingdomain of 4-1BB. Yet other aspects include methods of enhancing Tlymphocyte or natural killer (NK) cell activity in an individual andtreating an individual suffering from cancer by introducing into theindividual a T lymphocyte or NK cell comprising a chimeric receptor thatcomprises the signaling domain of 4-1BB. These aspects particularlyinclude the treatment of lung cancer, melanoma, breast cancer, prostatecancer, colon cancer, renal cell carcinoma, ovarian cancer,neuroblastoma, rhabdomyosarcoma, leukemia and lymphoma. Preferred cancertargets for use with the present invention are cancers of B cell origin,particularly including acute lymphoblastic leukemia, B-cell chroniclymphocytic leukemia and B-cell non-Hodgkin's lymphoma.

A different but related aspect of the present invention provides amethod for obtaining an enriched NK cell population suitable fortransduction with a chimeric receptor that comprises the signalingdomain of 4-1BB. This method comprises the expansion of NK cells withina mixed population of NK cells and T cells by co-culturing the mixedpopulation of cells with a cell line that activates NK cells and not Tlymphocytes. This NK activating cell line is composed of cells thatactivate NK cells, but not T lymphocytes, and which express membranebound interleukin-15 and a co-stimulatory factor ligand. In a particularembodiment the NK activating cell line is the K562 myeloid leukemia cellline or the Wilms tumor cell line HFWT. In another embodiment of theinvention the co-stimulatory factor ligand is CD137L.

Another aspect of the present invention is based on the concept thatexpression of chimeric receptors on NK cells could overcome HLA-mediatedinhibitory signals, thus endowing the cells with cytotoxicity againstotherwise NK-resistant cells. The invention provides a method thatallows specific and vigorous preferential expansion of NK cells lackingT-cell receptors (CD56⁺ CD3⁻ cells) and their highly efficienttransduction with chimeric receptors.

6. DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID No. 1 is the nucleotide sequence for human 4-1BB mRNA. The codingsequence for the human 4-1BB protein begins at position 129 and ends atposition 893.

SEQ ID No. 2 is the amino acid sequence of human 4-1BB. The signalingdomain begins at position 214 and ends at position 255.

SEQ. ID. No. 3 is the nucleotide sequence for murine 4-1BB mRNA. Thecoding sequence for the murine 4-1BB protein begins at position 146 andends at position 916.

SEQ ID. No. 4 is the amino acid sequence of murine 4-1BB. The signalingdomain begins at position 209 and ends at position 256.

7. DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of the CD19-truncated, CD19-ζ,CD19-28-ζ and CD19-BB-ζ receptor constructs.

FIG. 2 shows the percent of CD19-positive leukemia cell recovery in fourdifferent cell lines (380, 697, KOPN-57bi and OP-1) after 24 hours ofculture with NK cells with or without a chimeric receptor at a 1:1 ratiorelative to cultures with no NK cells. The bars represent each of the 4cell lines that are co-cultured with NK cells containing either “vector”which is MSCV-IRES GFP only; “trunc.” which is vector containingtruncated anti-CD19; “ζ” which is vector containing anti-CD19- CD3ζ; “28ζ” which is vector containing anti-CD19-CD28α-CD3ζ; or “BB-ζ” which isvector containing anti-CD19-4-1BB intracellular domain-CD3ζ. This figureshows that chimeric receptors confer anti-ALL activity to NK cells whichis improved by the addition of the co-stimulatory molecules CD28 or4-1BB.

8. DETAILED DESCRIPTION OF THE INVENTION Definitions

4-1BB: The term “4-1BB” refers to a membrane receptor protein alsotermed CD137, which is a member of the tumor necrosis factor receptor(TNFR) superfamily expressed on the surface of activated T-cells as atype of accessory molecule [Kwon et al., Proc. Natl. Acad. Sci. USA86:1963 (1989); Pollok et al., J. Immunol. 151:771 (1993)]. 4-1BB has amolecular weight of 55 kDa, and is found as a homodimer. It has beensuggested that 4-1BB mediates a signal transduction pathway from outsideof the cell to inside [Kim et al., J. Immunol. 151:1255 (1993)].

A human 4-1BB gene (SEQ ID NO:1) was isolated from a cDNA library madefrom activated human peripheral T-cell mRNA [Goodwin et al., Eur. J.Immunol. 23:2631 (1993);]. The amino acid sequence of human 4-1BB (SEQID NO: 2) shows 60% homology to mouse 4-1BB (SEQ ID NO:4)[Kwon et al.,Proc. Natl. Acad. Sci. USA 86:1963 (1989); Gen Bank No: NM_(—)011612]which indicates that the sequences are highly conserved. As mentionedsupra, 4-1BB belongs to the TNFR superfamily, along with CD40, CD27,TNFR-I, TNFR-II, Fas, and CD30 [Alderson et al., Eur. J. Immunol.24:2219 (1994)]. When a monoclonal antibody is bound to 4-1BB expressedon the surface of mouse T-cells, anti-CD3 T-cell activation is increasedmany fold [Pollok et al., J. Immunol. 150:771 (1993)].

4-1BB binds to a high-affinity ligand (4-1BB, also termed CD137L)expressed on several antigen-presenting cells such as macrophages andactivated B cells [Pollok et al., J. Immunol. 150:771 (1993) Schwarz etal., Blood 85:1043 (1995)). The interaction of 4-1BB and its ligandprovides a co-stimulatory signal leading to T cell activation and growth[Goodwin et al., Eur. J. Immunol. 23:2631 (1993); Alderson et al., Eur.J. Immunol. 24:2219 (1994); Hurtado et al., J. Immunol. 155:3360 (1995);Pollock et al., Eur. J. Immunol. 25:488 (1995); DeBenedette et al., J.Exp. Med. 181:985 (1995)]. These observations suggest an important rolefor 4-1BB in the regulation of T cell-mediated immune responses [Ignacioet al., Nature Med. 3:682 (1997)].

4-1BB ligand (CD137L) is claimed and described in U.S. Pat. No.5,674,704.

The term IL-15 (interleukin 15) refers to a cytokine that stimulates NKcells [Fehniger T A, Caligiuri M A. Blood 97(1):14-32 (2001)]. It hasbecome apparent that IL-15 presented through cell-to-cell contact has ahigher NK stimulating activity than soluble IL-15 [Dubois S, et al.,Immunity 17(5):537-547 (2002); Kobayashi H, et al., Blood (2004) PMID:15367431; Koka R, et al., J Immunol 173(6):3594-3598 (2004); Burkett PR,et al., J Exp Med 200(7):825-834 (2004)]. To express membrane-boundIL-15 a construct consisting of human IL-15 mature peptide (NM 172174)was fused to the signal peptide and transmembrane domain of human CD8α.

To specifically or preferentially expand NK cells means to culture amixed population of cells that contains a small number of NK cells sothat the NK cells proliferate to numbers greater than other cell typesin the population.

To activate T cells and NK cells means to induce a change in theirbiologic state by which the cells express activation markers, producecytokines, proliferate and/or become cytotoxic to target cells. Allthese changes can be produced by primary stimulatory signals.Co-stimulatory signals amplify the magnitude of the primary signals andsuppress cell death following initial stimulation resulting in a moredurable activation state and thus a higher cytotoxic capacity.

The terms T-cell and T lymphocyte are interchangeable and usedsynonymously herein.

The term “chimeric receptor” as used herein is defined as a cell-surfacereceptor comprising an extracellular ligand binding domain, atransmembrane domain and a cytoplasmic co-stimulatory signaling domainin a combination that is not naturally found together on a singleprotein. This particularly includes receptors wherein the extracellulardomain and the cytoplasmic domain are not naturally found together on asingle receptor protein. The chimeric receptors of the present inventionare intended primarily for use with T cells and natural killer (NK)cells.

The term “host cell” means any cell of any organism that is selected,modified, transformed, grown, used or manipulated in any way, for theproduction of a substance by the cell, for example the expression by thecell of a gene, a DNA or RNA sequence, a protein or an enzyme. Hostcells of the present invention include T cells and NK cells that containthe DNA or RNA sequences encoding the chimeric receptor and express thechimeric receptor on the cell surface. Host cells may be used forenhancing T lymphocyte activity, NK cell activity, treatment of cancer,and treatment of autoimmune diseases.

The terms “express” and “expression” mean allowing or causing theinformation in a gene or DNA sequence to become manifest, for exampleproducing a protein by activating the cellular functions involved intranscription and translation of a corresponding gene or DNA sequence. ADNA sequence is expressed in or by a cell to form an “expressionproduct” such as a protein. The expression product itself, e.g. theresulting protein, may also be said to be “expressed” by the cell. Anexpression product can be characterized as intracellular, extracellularor transmembrane. The term “intracellular” means something that isinside a cell. The term “extracellular” means something that is outsidea cell. The term transmembrane means something that has an extracellulardomain outside the cell, a portion embedded in the cell membrane and anintracellular domain inside the cell.

The term “transfection” means the introduction of a foreign nucleic acidinto a cell using recombinant DNA technology. The term “transformation”means the introduction of a “foreign” (i.e. extrinsic or extracellular)gene, DNA or RNA sequence to a host cell, so that the host cell willexpress the introduced gene or sequence to produce a desired substance,typically a protein or enzyme coded by the introduced gene or sequence.The introduced gene or sequence may also be called a “cloned” or“foreign” gene or sequence, may include regulatory or control sequences,such as start, stop, promoter, signal, secretion, or other sequencesused by a cell's genetic machinery. The gene or sequence may includenonfunctional sequences or sequences with no known function. A host cellthat receives and expresses introduced DNA or RNA has been “transformed”and is a “transformant” or a “clone.” The DNA or RNA introduced to ahost cell can come from any source, including cells of the same genus orspecies as the host cell, or cells of a different genus or species.

The term “transduction” means the introduction of a foreign nucleic acidinto a cell using a viral vector.

The terms “vector”, “cloning vector” and “expression vector” mean thevehicle by which a DNA or RNA sequence (e.g. a foreign gene) can beintroduced into a host cell, so as to transform the host and promoteexpression (e.g. transcription and translation) of the introducedsequence. Vectors include plasmids, phages, viruses, etc.

A solid support means any surface capable of having an agent attachedthereto and includes, without limitation, metals, glass, plastics,polymers, particles, microparticles, co-polymers, colloids, lipids,lipid bilayers, cell surfaces and the like. Essentially any surface thatis capable of retaining an agent bound or attached thereto. Aprototypical example of a solid support used herein, is a particle suchas a bead.

The term “substantially free of” means a population of cells, e.g. NKcells, that is at least 50% free of non-NK cells, or in certainembodiments at least 60, 70, 80, 85, or 90% free of non-NK cells.

A “co-stimulatory signal” refers to a signal, which in combination witha primary signal, such as TCR/CD3 ligation, leads to NK cellproliferation and/or upregulation or downregulation of key molecules.

Description of the Invention

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook et al, “Molecular Cloning:A Laboratory Manual” (1989); “Current Protocols in Molecular Biology”Volumes [-III [Ausubel, R. M., ed. (1994)]; “Cell Biology: A LaboratoryHandbook” Volumes I-III [J. E. Celis, ed. (1994))]; “Current Protocolsin Immunology” Volumes I-III [Coligan, J. E., ed. (1994)];“Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” [B. D. Haines & S. J. Higgins eds. (1985)];“Transcription And Translation” [B. D. Haines & S. J. Higgins, eds.(1984)]; “Animal CellCulture” [R.I. Freshney, ed. (1986)]; “ImmobilizedCells And Enzymes” [IRL Press, (1986)]; B. Perbal, “A Practical Guide ToMolecular Cloning” (1984); CURRENT PROTOCOLS IN IMMUNOLOGY Q. E.Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W. Strober,eds., 1991); ANNUAL REVIEW OF IMMUNOLOGY; as well as monographs injournals such as ADVANCES IN IMMUNOLOGY. All patents, patentapplications, and publications mentioned herein are hereby incorporatedherein by reference.

Primary T cells expressing chimeric receptors specific for tumor orviral antigens have considerable therapeutic potential as immunotherapyreagents. Unfortunately, their clinical value is limited by their rapidloss of function and failure to expand in vivo, presumably due to thelack of co-stimulator molecules on tumor cells and the inherentlimitations of signaling exclusively through the chimeric receptor.

The chimeric receptors of the present invention overcome this limitationwherein they have the capacity to provide both the primary effectoractivity and the co-stimulatory activity upon binding of the receptor toa single ligand. For instance, binding of the anti-CD 19-BB-ζ receptorto the CD19 ligand provides not only the primary effector function, butalso a proliferative and cytolytic effect.

T cells transduced with anti-CD19 chimeric receptors of the presentinvention which contain co-stimulatory molecules have remarkableanti-ALL capacity. However, the use of allogenic receptor-modified Tcells after hematopoietic cell transplantation might carry the risk ofsevere graft-versus-host disease (GvHD). In this setting, the use ofCD3-negative NK cells is attractive because they are not expectedtocause GvHD.

Studies suggest an anti-tumor effect of NK cells and Zeis et al., Br JHaematol 96: 757-61 (1997) have shown in mice that NK cells contributeto a graft-versus-leukemia effect, without inducing GvHD.

Expanding NK cells which can then be transfected with chimeric receptorsaccording to this method represents another aspect of the presentinvention.

The chimeric receptors of the present invention comprise anextracellular domain, a transmembrane domain and a cytoplasmic domain.The extracellular domain and transmembrane domain can be derived fromany desired source for such domains.

As described in U.S. Pat. Nos. 5,359,046, 5,686,281 and 6,103,521, theextracellular domain may be obtained from any of the wide variety ofextracellular domains or secreted proteins associated with ligandbinding and/or signal transduction. The extracellular domain may be partof a protein which is monomeric, homodimeric, heterodimeric, orassociated with a larger number of proteins in a non-covalent complex.In particular, the extracellular domain may consist of an Ig heavy chainwhich may in turn be covalently associated with Ig light chain by virtueof the presence of CH1 and hinge regions, or may become covalentlyassociated with other Ig heavy/light chain complexes by virtue of thepresence of hinge, CH2 and CH3 domains. In the latter case, theheavy/light chain complex that becomes joined to the chimeric constructmay constitute an antibody with a specificity distinct from the antibodyspecificity of the chimeric construct. Depending on the function of theantibody, the desired structure and the signal transduction, the entirechain may be used or a truncated chain may be used, where all or a partof the CH1, CH2, or CH3 domains may be removed or all or part of thehinge region may be removed.

Wherein an antitumor chimeric receptor is utilized, the tumor may be ofany kind as long as it has a cell surface antigen which may berecognized by the chimeric receptor. In a specific embodiment, thechimeric receptor may be for any cancer for which a specific monoclonalantibody exists or is capable of being generated. In particular, cancerssuch as neuroblastoma, small cell lung cancer, melanoma, ovarian cancer,renal cell carcinoma, colon cancer, Hodgkin's lymphoma, and childhoodacute lymphoblastic leukemia have antigens specific for the chimericreceptors.

The transmembrane domain may be contributed by the protein contributingthe multispecific extracellular inducer clustering domain, the proteincontributing the effector function signaling domain, the proteincontributing the proliferation signaling portion, or by a totallydifferent protein. For the most part it will be convenient to have thetransmembrane domain naturally associated with one of the domains. Insome cases it will be desirable to employ the transmembrane domain ofthe .zeta., .eta. or Fc.epsilon.R1.gamma. chains which contain acysteine residue capable of disulfide bonding, so that the resultingchimeric protein will be able to form disulfide linked dimers withitself, or with unmodified versions of the .zeta., .eta. orFc.epsilon.R1.gamma. chains or related proteins. In some instances, thetransmembrane domain will be selected or modified by amino acidsubstitution to avoid binding of such domains to the transmembranedomains of the same or different surface membrane proteins to minimizeinteractions with other members of the receptor complex. In other casesit will be desirable to employ the transmembrane domain of .zeta.,.eta., Fc.epsilon.R1-.gamma. and -.beta., MB1 (Ig.alpha.), B29 orCD3-.gamma., .zeta., or .epsilon., in order to retain physicalassociation with other members of the receptor complex.

The cytoplasmic domain of the chimeric receptors of the invention willcomprise the 4-1BB signaling domain by itself or combined with any otherdesired cytoplasmic domain(s) useful in the context of this chimericreceptor type. In a most preferred embodiment of the invention theextracellular domain comprises a single chain variable domain of ananti-CD 19 monoclonal antibody, the transmembrane domain comprises thehinge and transmembrane domain of CD8α, and the cytoplasmic domaincomprises the signaling domain of CD3ζ and the signaling domain of4-1BB. The extracellular domain of the preferred embodiment contains theanti-CD19 monoclonal antibody which is described in Nicholson IC, etal., Mol Immunol 34:1157-1165 (1997) plus the 21 amino acid signalpeptide of CD8α (translated from 63 nucleotides at positions 26-88 ofGenBank Accession No. NM_(—)001768). The CD8α hinge and transmembranedomain consists of 69 amino acids translated from the 207 nucleotides atpositions 815-1021 of GenBank Accession No. NM_(—)001768. The CD3ζsignaling domain of the preferred embodiment contains 112 amino acidstranslated from 339 nucleotides at positions 1022-1360 of GenBankAccession No. NM_(—)000734.

Antigen-specific cells can be expanded in vitro for use in adoptivecellular immunotherapy in which infusions of such cells have been shownto have anti-tumor reactivity in a tumor-bearing host. The compositionsand methods of this invention can be used to generate a population of Tlymphocyte or NK cells that deliver both primary and co-stimulatorysignals for use in immunotherapy in the treatment of cancer, inparticular the treatment of lung cancer, melanoma, breast cancer,prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer,neuroblastoma, rhabdomyosarcoma, leukemia and lymphoma.Immunotherapeutics, generally, rely on the use of immune effector cellsand molecules to target and destroy cancer cells. The effector may be alymphocyte carrying a surface molecule that interacts, either directlyor indirectly, with a tumor cell target. Various effector cells includecytotoxic T cells and NK cells. The compositions and methods describedin the present invention may be utilized in conjunction with other typesof therapy for cancer, such as chemotherapy, surgery, radiation, genetherapy, and so forth.

In adoptive immunotherapy, the patient's circulating lymphocytes, ortumor infiltrated lymphocytes, are isolated in vitro, activated bylymphokines such as IL-2 or transduced with genes for tumor necrosis,and readministered [Rosenberg et al., N. Engl. J. Med. 319:1767 (1988)].To achieve this, one would administer to an animal, or human patient, animmunologically effective amount of activated lymphocytes geneticallymodified to express a tumor-specific chimeric receptor gene as describedherein. The activated lymphocytes will most preferably be the patient'sown cells that were earlier isolated from a blood or tumor sample andactivated and expanded in vitro. In aspects of the present invention Tlymphocytes or NK cells from a patient having a cancer of B cell originsuch as lymphoblastic leukemia, B-cell chronic lymphocytic leukemia orB-cell non-Hodgkin's lymphoma would be isolated and tranduced with theCD19-BB-ζ polynucleotide so that a chimeric receptor containing 4-1BB inthe cytoplasmic domain is express on the cell surface of the T cell orNK cell. The modified cells would then be readministered into thepatient to target and kill the tumor cells.

As shown in one Example infra, primary T-cells were transduced with theanti-CD19-BB-ζ receptor of the present invention. One week aftertransduction the T-cells had expanded 3-4 fold in contrast to cells thatwere transduced with a chimeric receptor that lacked 4-1BB. After 3weeks in culture the T-cells had expanded by more than 16-fold.

T-cells that were transduced with the anti-CD19-BB-ζ receptor andcultured in 200 IU/mL of IL-2 for two weeks, then removed from IL-2 andexposed to irradiated OP-1 cells underwent apoptosis. However, cellscultured in 10 IU/mL of IL-2 and exposed to irradiated OP-1 cells fortwo weeks after transduction remained viable. T-cells that weretransduced with CD19 chimeric receptors that lacked 4-1BB underwentapoptosis under these same conditions. These results show that 4-1BBco-stimulation confers a survival advantage on lymphocytes, whichovercomes a major obstacle with current chimeric receptors used inimmunotherapy.

To determine if T-cells transduced with the anti-CD19-BB-ζ receptorexhibited cytotoxic activity under conditions necessary forimmunotherapy, their cytotoxic activity at low effector:target (E:T)ratios were measured. As described in the Example infra, T-cellstransduced with the anti-CD19-BB-ζ receptor and control vectors wereexpanded in vitro for two weeks and mixed with OP-1 cells at various E:Tratios (1:1, 0.1:1, and 0.01:1). Viable leukemic cells were countedafter one week of culture. T-cells expressing the anti-CD19-BB-ζreceptor exhibited cytotoxic activity at the 1:1 and 0.1:1 ratiosagainst all CD19⁺ cell lines tested. The anti-CD19-BB-ζ receptor was noteffective at the 0.01:1 ratio. The CD19 chimeric receptor that lacked4-1BB showed cytotoxic activity at the 1:1 ratio, but at the 0.1:1 ratiothe results were inferior to the anti-CD19-BB-ζ receptor.

A surprising result obtained with the anti-CD19-BB-ζ receptor was thatthe T-cells transduced with the receptor exhibited cytotoxic activitytoward CD 19⁺ leukemic cells at a ratio of 0.01:1 when the leukemiccells were co-cultured with bone marrow-derived mesenchymal cells. Thisresult shows that T-cells transduced with the anti-CD19-BB-ζ receptorexhibit cytotoxic activity in an environment critical for B-lineageleukemic cell growth. Another unexpected result was that expression ofthe anti-CD19-BB-ζ receptor caused higher levels of TRAIL stimulation.

Furthermore, IL-2, which causes CD8⁺ cells to expand more vigorously,levels in cells expressing the anti-CD19-BB-ζ receptor were higher thanin cells expressing the other receptors tested. These results furthersupport the use of the anti-CD19-BB-ζ receptor for immunotherapy.

Construction of the Anti-CD19-BB-ζ receptor

The present invention provides a chimeric receptor construct whichcontains the signaling domain of 4-1BB and fragments thereof. In apreferred embodiment of the invention, the genetic fragments used in thechimeric receptor were generated using splicing by overlapping extensionby PCR (SOE-PCR), a technique useful for generating hybrid proteins ofimmunological interest. [Warrens A N, et al. Gene 20;186: 29-35 (1997)].Other procedures used to generate the polynucleotides and vectorconstructs of the present invention are well known in the art.

Transduction of T-cells

As shown in the Examples, infra, a polynucleotide expressing a chimericreceptor capable of providing both primary effector and co-stimulatoryactivities was introduced into T-cells and NK cells via retroviraltransduction. References describing retroviral transduction of genes areAnderson et al., U.S. Pat. No. 5,399,346; Mann et al., Cell 33:153(1983); Temin et al., U.S. Pat. No. 4,650,764; Temin et al., U.S. Pat.No. 4,980,289; Markowitz et al., J. Virol. 62:1120 (1988); Temin et al.,U.S. Pat. No. 5,124,263; International Patent Publication No. WO95/07358, published Mar. 16, 1995, by Dougherty et al.; and Kuo et al.,Blood 82:845 (1993). International Patent Publication No. WO 95/07358describes high efficiency transduction of primary B lymphocytes.

Expansion of NK Cells

The present invention shows that human primary NK cells may be expandedin the presence of a myeloid cell line that has been geneticallymodified to express membrane bound IL-15 and 4-1BB ligand (CD137L). Acell line modified in this way which does not have MHC class I and IImolecules is highly susceptible to NK cell lysis and activates NK cells.

For example, K562 myeloid cells can be transduced with a chimericprotein construct consisting of human IL-15 mature peptide fused to thesignal peptide and transmembrane domain of human CD8α and GFP.Transduced cells can then be single-cell cloned by limiting dilution anda clone with the highest GFP expression and surface IL-15 selected. Thisclone can then be transduced with human CD137L, creating aK562-mb15-137L cell line.

To preferentially expand NK cells, peripheral blood mononuclear cellcultures containing NK cells are cultured with a K562-mb15-137L cellline in the presence of 10IU/mL of IL-2 for a period of time sufficientto activate and enrich for a population of NK cells. This period canrange from 2 to 20 days, preferably about 5 days. Expanded NK cells maythen be transduced with the anti-CD19-BB-c chimeric receptor.

Administration of Activated T Cells and NK Cells

Methods of re-introducing cellular components are known in the art andinclude procedures such as those exemplified in U.S. Pat. Nos. 4,844,893and 4,690,915. The amount of activated T cells or NK cells used can varybetween in vitro and in vivo uses, as well as with the amount and typeof the target cells. The amount administered will also vary depending onthe condition of the patient and should be determined by considering allappropriate factors by the practitioner.

Obtaining an enriched population of NK cells for use in therapy has beendifficult to achieve. Specific NK cell expansion has been problematic toachieve with established methods, where CD3+ T cells preferentiallyexpand. Even after T cell depletion, residual T cells typically becomeprominent after stimulation. However, in accordance with the teachingsof the present invention NK cells may be preferentially expanded byexposure to cells that lack or poorly express major histocompatibilitycomplex I and/or II molecules and which have been genetically modifiedto express membrane bound IL-15 and 4-1BB ligand (CDI37L). Such celllines include, but are not necessarily limited to, K562 [ATCC, CCL 243;Lozzio et al., Blood 45(3): 321-334 (1975); Klein et al., Int. J. Cancer18: 421-431 (1976)], and the Wilms tumor cell line HFWT. [Fehniger T A,Caligiuri M A. Int Rev Immunol 20(3-4):503-534 (2001); Harada H, et al.,Exp Hematol 32(7):614-621 (2004)], the uterine endometrium tumor cellline HHUA, the melanoma cell line HMV-II, the hepatoblastoma cell lineHuH-6, the lung small cell carcinoma cell lines Lu-130 and Lu-134-A, theneutoblastoma cell lines NB 19 and N1369, the embryonal carcinoma cellline from testis NEC 14, the cervix carcinoma cell line TCO-2, and thebone marrow-metastated neuroblastoma cell line TNB 1 [Harada H., et al.,Jpn. J. Cancer Res 93: 313-319 (2002)]. Preferably the cell line usedlacks or poorly expresses both MHC I and II molecules, such as the K562and HFWT cell lines.

A solid support may be used instead of a cell line. Such supports willhave attached on its surface at least one molecule capable of binding toNK cells and inducing a primary activation event and/or a proliferativeresponse or capable of binding a molecule having such an affect therebyacting as a scaffold. The support may have attached to its surface theCD137 ligand protein, a CD137 antibody, the IL-15 protein or an IL-15receptor antibody. Preferably, the support will have IL-15 receptorantibody and CD137 antibody bound on its surface.

The invention is intended to include the use of fragments, mutants, orvariants (e.g., modified forms) of the IL-15 and/or CD137 ligandproteins or antigens that retain the ability to induce stimulation andproliferation of NK cells. A “form of the protein” is intended to mean aprotein that shares a significant homology with the IL-15 or CD137ligand proteins or antigen and is capable of effecting stimulation andproliferation of NK cells. The terms “biologically active” or“biologically active form of the protein,” as used herein, are meant toinclude forms of the proteins or antigens that are capable of effectingenhanced activated NK cell proliferation. One skilled in the art canselect such forms based on their ability to enhance NK cell activationand proliferation upon introduction of a nucleic acid encoding saidproteins into a cell line. The ability of a specific form of the IL-15or CD137 ligand protein or antigen to enhance NK cell proliferation canbe readily determined, for example, by measuring cell proliferation oreffector function by any known assay or method.

Antigen-specific cells can be expanded in vitro for use in adoptivecellular immunotherapy in which infusions of such cells have been shownto have anti-tumor reactivity in a tumor-bearing host. The compositionsand methods of this invention can be used to generate a population of NKcells that deliver both primary and co-stimulatory signals for use inimmunotherapy in the treatment of cancer, in particular the treatment oflung cancer, melanoma, breast cancer, prostate cancer, colon cancer,renal cell carcinoma, ovarian cancer, neuroblastoma, rhabdomyosarcoma,leukemia and lymphoma. The compositions and methods described in thepresent invention may be utilized in conjunction with other types oftherapy for cancer, such as chemotherapy, surgery, radiation, genetherapy, and so forth.

9. EXAMPLES 9.1 Example 1 Introduction

In approximately 20% of children and 65% of adults with acutelymphoblastic leukemia (ALL), drug-resistant leukemic cells surviveintensive chemotherapy and cause disease recurrence. [Pui C H et al,Childhood acute lymphoblastic leukemia—Current status and futureperspectives. Lancet Oncology2:597-607 (2001); Verma A, Stock W.Management of adult acute lymphoblastic leukemia: moving toward arisk-adapted approach. Curr Opin Oncol 13:14-20T (2001)]lymphocyte-based cell therapy should bypass cellular mechanisms of drugresistance. Its potential clinical value for leukemia is demonstrated bythe association between T-cell-mediated graft-versus-host disease (GvHD)and delay or suppression of leukemia recurrence after allogeneic stemcell transplantation. [Champlin R. T-cell depletion to preventgraft-versus-host disease after bone marrow transplantation. HematolOncol Clin North Am 4:687-698 (1990); Porter D L, Antin J H. Thegraft-versus-leukemia effects of allogeneic cell therapy. Annu Rev Med50:369-86.:369-386 (1999); Appelbaum F R. Haematopoietic celltransplantation as immunotherapy. Nature 411:385-389 (2001)]Manipulation of GvHD by infusion of donor lymphocytes can produce ameasurable anti-leukemic effect. [Porter D L, et al. Induction ofgraft-versus-host disease as immunotherapy for relapsed chronic myeloidleukemia. N Engl J Med 330:100-106 (1994); Kolb H J, et al.Graft-versus-leukemia effect of donor lymphocyte transfusions in marrowgrafted patients. Blood 6:2041-2050 (1995); Slavin S, et al. Allogeneiccell therapy with donor peripheral blood cells and recombinant humaninterleukin-2 to treat leukemia relapse after allogeneic bone marrowtransplantation. Blood 87:2195-2204 (1996); Collins R H, et al. Donorleukocyte infusions in 140 patients with relapsed malignancy afterallogeneic bone marrow transplantation. J Clin Oncol 15:433-444 (1997)]However, in patients with ALL this effect is often limited, [Kolb H J,et al. Graft-versus-leukemia effect of donor lymphocyte transfusions inmarrow grafted patients. Blood 86:2041-2050 (1995); Verdonck L F, et al.Donor leukocyte infusions for recurrent hematologic malignancies afterallogeneic bone marrow transplantation: impact of infused and residualdonor T cells. Bone Marrow Transplant 22:1057-1063 (1998); Collins R H,Jr., et al. Donor leukocyte infusions in acute lymphocytic leukemia.Bone Marrow Transplant 26:511-516 (2000)] possibly reflecting inadequateT-cell stimulation by leukemic lymphoblasts.

T lymphocyte specificity can be redirected through expression ofchimeric immune receptors consisting of an extracellularantibody-derived single-chain variable domain (scFv) and anintracellular signal transduction molecule (e.g., the signaling domainof CD3ζ or FcγRIII). [Geiger T L, Jyothi M D. Development andapplication of receptor-modified T lymphocytes for adoptiveimmunotherapy. Transfus Med Rev 15:21-34 (2001); Schumacher TN.T-cell-receptor gene therapy. Nat Rev Immunol. 2:512-519 (2002);Sadelain M, et al. Targeting tumours with genetically enhanced Tlymphocytes. Nat Rev Cancer 3:35-45 (2003)] Such T lymphocytes can beactivated by cell surface epitopes targeted by the scFv and can kill theepitope-presenting cells. The first requirement to redirect T cellsagainst ALL cells is the identification of target molecules that areselectively expressed by leukemic cells. In B-lineage ALL, CD19 is anattractive target, because it is expressed on virtually all leukemiclymphoblasts in almost all cases. [Campana D, Behm F G.Immunophenotyping of leukemia. J Immunol Methods 243:59-75 (2000)] It isnot expressed by normal non-hematopoietic tissues, and amonghematopoietic cells, it is expressed only by B-lineage lymphoid cells.[Campana D, Behm F G. Immunophenotyping of leukemia. J Immunol Methods243:59-75 (2000); Nadler L M, et al. B4, a human B lymphocyte-associatedantigen expressed on normal, mitogen-activated, and malignant Blymphocytes. J Immunol 131:244-250 (1983)] Recent studies have shownthat T-cells expressing anti-CD19 scFv and CD3C signaling domain canproliferate when mixed with CD19⁺ cells and can lyse CD19⁺ target cells.[Cooper L J, et al. T-cell clones can be rendered specific for CD19:toward the selective augmentation of the graft-versus-B-lineage leukemiaeffect. Blood 101:1637-1644 (2003); Brentjens R J, et al. Eradication ofsystemic B-cell tumors by genetically targeted human T lymphocytesco-stimulated by CD80 and interleukin-15. Nat Med 9:279-286 (2003)]

A prerequisite for the success of T-cell therapy is the capacity of theengineered T lymphocytes to expand and produce a vigorous and durableanti-leukemic response in vivo. The engagement of the TCR, althoughnecessary, is not sufficient to fully activate T cells; a second signal,or co-stimulus, is also required. [Liebowitz D N, et al. Costimulatoryapproaches to adoptive immunotherapy. Curr Opin Oncol 10:533-541 (1998);Allison J P, Lanier L L. Structure, function, and serology of the T-cellantigen receptor complex. Annu Rev Immunol 5:503-540 (1987); Salomon B,Bluestone J A. Complexities of CD28/B7: CTLA-4 costimulatory pathways inautoimmunity and transplantation. Annu Rev Immunol 19:225-52.:225-252(2001)] This could be a major obstacle for chimeric receptor-basedtherapy of B-lineage ALL, because B-lineage leukemic lymphoblastsgenerally lack B7 molecules that bind to CD28 on T-lymphocytes andtrigger the CD28-mediated co-stimulatory pathway. [Cardoso A A, et al.Pre-B acute lymphoblastic leukemia cells may induce T-cell anergy toalloantigen. Blood 88:41-48 (1996)] This limitation might be overcome byincorporating the signal transduction domain of CD28 into chimericreceptors. [Eshhar Z, et al. Functional expression of chimeric receptorgenes in human T cells. J Immunol Methods 2001;248:67-76 (2001); HombachA, et al. Tumor-specific T cell activation by recombinantimmunoreceptors: CD3 zeta signaling and CD28 costimulation aresimultaneously required for efficient IL-2 secretion and can beintegrated into one combined CD28/CD3 zeta signaling receptor molecule.J Immunol 167:6123-6131 (2001); Geiger T L, et al. Integrated src kinaseand costimulatory activity enhances signal transduction throughsingle-chain chimeric receptors in T lymphocytes. Blood 98:2364-2371(2001); Maher J, et al. Human T-lymphocyte cytotoxicity andproliferation directed by a single chimeric TCRzeta /CD28 receptor. NatBiotechnol 20:70-75 (2002)] Murine T cells bearing such receptors haveshown a greater capacity to inhibit cancer cell growth and metastasis inmice than those with chimeric receptors lacking this domain. [Haynes NM, et al. Rejection of syngeneic colon carcinoma by CTLs expressingsingle-chain antibody receptors codelivering CD28 costimulation. JImmunol 169:5780-5786 (2002); Haynes N M, et al. Single-chain antigenrecognition receptors that costimulate potent rejection of establishedexperimental tumors. Blood 100:3155-3163 (2002)]

A second co-stimulatory pathway in T cells, independent of CD28signaling, is mediated by 4-1BB (CD137), a member of the tumor necrosisfactor (TNF) receptor family. [Sica G, Chen L. Modulation of the immuneresponse through 4-1BB. In: Habib N, ed. Cancer gene therapy: pastachievements and future challenges. New York: Kluwer Academic/PlenumPublishers; 355-362 (2000)] 4-1BB stimulation significantly enhancessurvival and clonal expansion of CD8+ T-lymphocytes, and CD8+ T-cellresponses in a variety of settings, including viral infection, allograftrejection, and tumor immunity. [Shuford WW, et al. 4-1BB costimulatorysignals preferentially induce CD8+T cell proliferation and lead to theamplification in vivo of cytotoxic T cell responses. J Exp Med 186:47-55(1997); Melero I, et al. Monoclonal antibodies against the 4-1BB T-cellactivation molecule eradicate established tumors. Nat Med 3:682-685(1997); Melero I, et al. Amplification of tumor immunity by genetransfer of the co-stimulatory 4-1BB ligand: synergy with the CD28co-stimulatory pathway. Eur J Immunol 28:1116-1121 (1998); Takahashi C,et al. Cutting edge: 4-1BB is a bona fide CD8 T cell survival signal. JImmunol 162:5037-5040 (1999); Martinet O, et al. T cell activation withsystemic agonistic antibody versus local 4-1BB ligand gene deliverycombined with interleukin-12 eradicate liver metastases of breastcancer. Gene Ther 9:786-792 (2002); May K F, Jr., et al. Anti-4-1BBmonoclonal antibody enhances rejection of large tumor burden bypromoting survival but not clonal expansion of tumor-specific CD8+ Tcells. Cancer Res 62:3459-3465 (2002)] However, the natural ligand of4-1BB is weakly and heterogeneously expressed in B-lineage ALL cells (C.Imai, D. Campana, unpublished observations). Therefore, it is likelythat this important co-stimulatory signal, like CD28, can becomeoperational only if 4-1BB is added to chimeric receptors. However, it isnot known whether such receptors would help deliver effective T-cellresponses to cancer cells and, if so, whether these would be equivalentto those elicited by receptors containing CD28.

We constructed a chimeric T-cell receptor specific for CD19 thatcontains a 4-1BB signaling domain. We determined whether T cellstransduced with these receptors could effectively destroy B-lineage ALLcell lines and primary leukemic cells under culture conditions thatapproximate the in vivo microenvironment where leukemic cells grow. Wecompared the properties of T-cells expressing the 4-1BB-containingreceptor to those of T-cells expressing an equivalent receptor lacking4-1BB or containing CD28 instead.

Materials And Methods

Cells

Available in our laboratory were the human B-lineage ALL cell line OP-1,developed from the primary leukemic cells of a patient with newlydiagnosed B-lineage ALL with the t(9;22)(q34;q11) karyotype and theBCR-ABL gene fusion; [Manabe A, et al. Interleukin-4 induces programmedcell death (apoptosis) in cases of high-risk acute lymphoblasticleukemia. Blood 83:1731-1737 (1994)] the B-lineage ALL cell linesRS4;11, [Stong R C, et al. Human acute leukemia cell line with thet(4;11) chromosomal rearrangement exhibits B lineage and monocyticcharacteristics. Blood 1985;65:21-31 (1985)] and REH [Rosenfeld C, etal. Phenotypic characterisation of a unique non-T, non-B acutelymphoblastic leukaemia cell line. Nature 267:841-843 (1977)]; theT-cell lines Jurkat [Schneider U, et al. Characterization of EBV-genomenegative “null” and “T” cell lines derived from children with acutelymphoblastic leukemia and leukemic transformed non-Hodgkin lymphoma.Int J Cancer 1977;19:621-626 (1977)] and CEM-C7 [Harmon J M, et al.Dexamethasone induces irreversible G1 arrest and death of a humanlymphoid cell line. J Cell Physiol 98:267-278 (1979)]; and the myeloidcell lines K562 [Koeffler H P, Golde D W. Acute myelogenous leukemia: ahuman cell line responsive to colony-stimulating activity. Science200:1153-1154 (1978)] and U-937. [Sundstrom C, Nilsson K. Establishmentand characterization of a human histiocytic lymphoma cell line (U-937).Int J Cancer 1976;17:565-577 (1976)] Cells were maintained in RPMI-1640(Gibco, Grand Island, N.Y.) with 10% fetal calf serum (FCS;BioWhittaker, Walkersville, Md.) and antibiotics. Human adenocarcinomaHeLa cells and embryonic kidney fibroblast 293T cells, maintained inDMEM (MediaTech, Herndon, Va.) supplemented with 10% FCS andantibiotics, were also used.

We used primary leukemia cells obtained from 5 patients with newlydiagnosed B-lineage ALL with the approval of the St. Jude Children'sResearch Hospital Institutional Review Board and with appropriateinformed consent. The diagnosis of B-lineage ALL was unequivocal bymorphologic, cytochemical, and immunophenotypic criteria; in each case,more than 95% of leukemic cells were positive for CD 19. Peripheralblood samples were obtained from 7 healthy adult donors. Mononuclearcells were collected from the samples by centrifugation on a Lymphoprepdensity step (Nycomed, Oslo, Norway) and were washed two times inphosphate-buffered saline (PBS) and once in AIM-V medium (Gibco).

Plasmids

The plasmid encoding anti-CD19 scFv was obtained from Dr. I. Nicholson(Child Health Research Institute, Adelaide, Australia). [Nicholson I C,et al. Construction and characterisation of a functional CD19 specificsingle chain Fv fragment for immunotherapy of B lineage leukaemia andlymphoma. Mol Immunol 34:1157-1165 (1997)] The pMSCV-IRES-GFP,pEQPAM3(-E), and pRDF were obtained from Dr. E. Vanin at ourinstitution. Signal peptide, hinge and transmembrane domain of CD8a, andintracellular domains of 4-1BB, CD28, CD3ζ and CD19 were subcloned bypolymerase chain reaction (PCR) using a human spleen cDNA library (fromDr. G. Neale, St. Jude Children's Research Hospital) as a template. FIG.1 shows a schematic representation of the anti-CD19-ζ, anti-CD19-BB-ζ,anti-CD19-28-ζ and anti-CD19-truncated (control) constructs. We usedsplicing by overlapping extension by PCR (SOE-PCR) to assemble severalgenetic fragments. [Warrens A N, et al. Splicing by overlap extension byPCR using asymmetric amplification: an improved technique for thegeneration of hybrid proteins of immunological interest. Gene20;186:29-35 (1997)] The sequence of each genetic fragment was confirmedby direct sequencing. The resulting expression cassettes were subclonedinto EcoRI and XhoI sites of MSCV-IRES-GFP.

To transduce CD19-negative K562 cells with CD19, we constructed aMSCV-IRES-DsRed vector. The IRES and DsRed sequences were subcloned fromMSCV-IRES-GFP and pDsRedN1 (Clontech, Palo Alto, Calif.), respectively,and assembled by SOE-PCR. The IRES-DsRed cassette was digested andligated into XhoI and NotI sites of MSCV-IRES-GFP. The expressioncassette for CD19 was subsequently ligated into EcoRI and XhoI sites ofMSCV-IRES-DsRed vector.

Virus Production and Gene Transduction

To generate RD114-pseudotyped retrovirus, we used calcium phosphate DNAprecipitation to transfect 3×10⁶ 293T cells, maintained in 10-cm tissueculture dishes (Falcon, Becton Dickinson, Franklin Lakes, N.J.) for 24hours, with 8 μg of one of the vectors anti-CD19-ζ, anti-CD19-BB-ζ,anti-CD19-28-ζ or anti-CD19-truncated, 8 μg of pEQ-PAM3(-E) and 4 μg ofpRDF. After 24 hours, medium was replaced with RPMI-1640 with 10% FCSand antibiotics. Conditioned medium containing retrovirus was harvested48 hours and 72 hours after transfection, immediately frozen in dry ice,and stored at −80° C. until use. HeLa cells were used to titrate virusconcentration.

Peripheral blood mononuclear cells were incubated in a tissue culturedish for 2 hours to remove adherent cells. Non-adherent cells werecollected and prestimulated for 48 hours with 7 μg/mL PHA-M (Sigma, St.Louis, Mo.) and 200 IU/mL human IL-2 (National Cancer Institute BRBPreclinical Repository, Rockville, Md.) in RPMI-1640 and 10% FCS. Cellswere then transduced as follows. A 14-mL polypropylene centrifuge tube(Falcon) was coated with 0.5 mL of human fibronectin (Sigma) diluted to100 μg/mL for 2 hours at room temperature and then incubated with 2%bovine serum albumin (Sigma) for 30 minutes. Prestimulated cells (2×10⁵)were resuspended in the fibronectin-coated tube in 2-3 mL ofvirus-conditioned medium with polybrene (4 μg/mL; Sigma) and centrifugedat 2400×g for 2 hours. The multiplicity of infection (4 to 8) wasidentical in each experiment comparing the activity of differentchimeric receptors. After centrifugation, cells were left undisturbedfor 24 hours in a humidified incubator at 37° C., 5% CO₂. Thetransduction procedure was repeated on two successive days. Cells werethen washed twice with RPMI-1640 and maintained in RPMI-1640, 10% FCS,and 200 IU/mL of IL-2 until use.

A similar procedure was used to express chimeric receptors in Jurkatcells, except that cells were not prestimulated. K562 cells expressingCD19 were created by resuspending 2×10⁵ K562 cells in 3 mL ofMSCV-CD19-IRES-DsRed virus medium with 4 μg/mL polybrene in afibronectin-coated tube; the tube was centrifuged at 2400×g for 2 hoursand left undisturbed in an incubator for 24 hours. Control cells weretransduced with the vector only. These procedures were repeated on 3successive days. After confirming CD19 and DsRed expression, cells weresubjected to single-cell sorting with a fluorescence-activated cellsorter (MoFlo, Cytomation, Fort Collins, Colo.). The clones that showedthe highest expression of DsRed and CD19 and of DsRed alone wereselected for further experiments.

Detection of Chimeric Receptor Expression

Transduced Jurkat and peripheral blood cells were stained with goatanti-mouse (Fab)2 polyclonal antibody conjugated with biotin (JacksonImmunoresearch, West Grove, Pa.) followed by streptavidin conjugated toperidinin chlorophyll protein (PerCP; Becton Dickinson, San Jose,Calif.). Patterns of CD4, CD8, and CD28 expression were also analyzed byusing anti-CD4 and anti-CD28 conjugated to PE and anti-CD8 conjugated toPerCP (antibodies from Becton Dickinson, and Pharmingen, San Diego,Calif.). Antibody staining was detected with a FACScan flow cytometer(Becton Dickinson).

For Western blotting, 2×10⁷ cells were lysed in 1 mL RIPA buffer (PBS,1% Triton-X100, 0.5% sodium deoxycholate, 0.1% SDS) containing 3 μg/mLof pepstatin, 3 μg/mL of leupeptin, 1 mM of PMSF, 2mM of EDTA, and 5μg/mL of aprotinin. Centrifuged lysate supernatants were boiled with anequal volume of loading buffer with or without 0.1 M DTT, then wereseparated by SDS-PAGE on a precast 12% acrylamide gel (BioRad, Hercules,Calif.). The proteins were transferred to a PVDF membrane, which wasincubated with primary mouse anti-human CD3ζ monoclonal antibody (clone8D3; Pharmingen), 1 μg/mL for 12 hours at 4° C. Membranes were thenwashed, incubated with a 1:500 dilution of goat anti-mouse IgGhorseradish peroxidase-conjugated second antibody for 1 hour, anddeveloped by using the ECP kit (Pharmacia, Piscataway, N.J.).

Changes in Gene Expression and Cytokine Production after ReceptorLigation

Jurkat cells transduced with the chimeric receptors were cocultured withOP-1 leukemic cells fixed with 0.5% paraformaldehyde at an effector :target (E : T) ratio of 1:1. RNA was extracted using Trizol Reagent(Invitrogen, Carlsbad, Calif.). Gene expression of Jurkat cells wasanalyzed using HG-U133A GeneChip microarrays (Affymetrix, Santa Clara,Calif.) as previously described. [Yeoh E J, et al. Classification,subtype discovery, and prediction of outcome in pediatric acutelymphoblastic leukemia by gene expression profiling. Cancer Cell2002;1:133-143 (2002); Ross M E, et al. Classification of pediatricacute lymphoblastic leukemia by gene expression profiling. Blood. May2003; 10.1182/blood-2003-01-0338 (2003)] Arrays were scanned using alaser confocal scanner (Agilent, Palo Alto, Calif.) and analyzed withAffymetrix Microarray suite 5.0. We used an arbitrary factor of 2 orhigher to define gene overexpression. IL-2, TNF-relatedapoptosis-inducing ligand (TRAIL), OX40, IL-3 and β-actin transcriptswere detected by semi-quantitative reverse transcriptase-polymerasechain reaction (RT-PCR) using Jurkat cells stimulated as above; primerswere designed using the Primer3 software developed by the WhiteheadInstitute for Biomedical Research.

For cytokine production, Jurkat cells and primary lymphocytes (2×10⁵ in200 μl) expressing chimeric receptors were stimulated with OP-1 cells ata 1:1 E:T ratio for 24 hours. Levels of IL-2 and IFNγ in culturesupernatants were determined with a Bio-Plex assay (BioRad). Lymphocytesbefore and after stimulation were also labeled with anti-TRAIL-PE(Becton Dickinson).

Expansion and Purification of Receptor-Transduced Primary T Cells

Receptor-transduced lymphocytes (3×10⁵) were co-cultured with 1.5×10⁵irradiated OP-1 cells in RPMI-1640 with 10% FCS with or withoutexogenous IL-2. Cells were pulsed weekly with irradiated target cells atan E:T ratio of 2:1. Cells were counted by Trypan-blue dye exclusion andby flow cytometry to confirm the presence of GFP-positive cells and theabsence of CD19-positive cells. To prepare pure populations of CD8⁺cells expressing chimeric receptors, we labeled cells with aPE-conjugated anti-CD8 antibody (Becton Dickinson) that had beenpreviously dialyzed to remove preservatives and then sterile-filtered.CD8⁺ GFP+ cells were isolated using a fluorescence-activated cell sorter(MoFlo).

Cytotoxicity Assays

The cytolytic activity of transductants was measured by assays oflactate dehydrogenase (LDH) release using the Cytotoxicity Detection Kit(Roche, Indianapolis, Ind.) according to the manufacturer'sinstructions. Briefly, 2×10⁴ target cells were placed in 96-wellV-bottom tissue culture plates (Costar, Cambridge, Mass.) and coculturedin triplicate in RPMI-1640 supplemented with 1% FCS, with primarylymphocytes transduced with chimeric receptors. After 5 hours, cell-freesupernatant was harvested and immediately analyzed for LDH activity.Percent specific cytolysis was calculated by using the formula:(Test−effector control−low control/high control−low control)×100, inwhich “high control” is the value obtained from supernatant of targetcells exposed to 1% Triton-X-100, “effector control” is the spontaneousLDH release value of lymphocytes alone, “low control” is the spontaneousLDH release value of target cells alone; background control (the valueobtained from medium alone) was subtracted from each value before thecalculation.

The anti-leukemic activity of receptor-transduced lymphocytes was alsoassessed in 7-day cultures using lower E:T ratios. For this purpose, weused bone marrow—derived mesenchymal cells to support the viability ofleukemic cells. [Nishigaki H, et al. Prevalence and growthcharacteristics of malignant stem cells in B-lineage acute lymphoblasticleukemia. Blood 89:3735-3744 (1997); Mihara K, et al. Development andfunctional characterization of human bone marrow mesenchymal cellsimmortalized by enforced expression of telomerase. Br J Haematol120:846-849 (2003)] Briefly, 2×10⁴ human mesenchymal cells immortalizedby enforced expression of telomerase reverse transcriptase were platedon a 96-well tissue culture plate precoated with 1% gelatin. After 5days, 1×10⁴ CD19+ target cells (in case of cell lines) or 2×10⁵ CD 19+target cells (in case of primary ALL cells) were plated on the wells andallowed to rest for 2 hours. After extensive washing to remove residualIL-2-containing medium, receptor-transduced primary T cells were addedto the wells at the proportion indicated in Results. Cultures wereperformed in the absence of exogenous IL-2. Plates were incubated at 37°C. in 5% CO2 for 5-7 days. Cells were harvested, passed through a19-gauge needle to disrupt residual mesenchymal-cell aggregates, stainedwith anti-CD 19-PE antibody, and assayed by flow cytometry as previouslydescribed. [Ito C, et al. Hyperdiploid acute lymphoblastic leukemia with51 to 65 chromosomes: A distinct biological entity with a markedpropensity to undergo apoptosis. Blood 93:315-320 (1999); SrivannaboonK, et al. Interleukin-4 variant (BAY 36-1677) selectively inducesapoptosis in acute lymphoblastic leukemia cells. Blood 97:752-758(2001)] Expression of DsRed served as a marker of residual K562 cells.Experiments were done in triplicate.

Results

Transduction of Primary Human T Lymphocytes with Anti-CD19-BB-ζ ChimericReceptors

In preliminary experiments, transduction of lymphocytes stimulated withPHA (7 μg/mL) and IL-2 (200 IU/mL) for 48 hours, followed bycentrifugation (at 240033 g) of the activated lymphocytes withretroviral supernatant in tubes coated with fibronectin, consistentlyyielded a high percentage of chimeric receptor and GFP expression; thismethod was used in all subsequent experiments. In 75 transductionexperiments, 31% to 86% (median, 64%) of mononuclear cells expressedGFP. In experiments with cells obtained from 6 donors, we tested theimmunophenotype of the cells transduced with anti-CD19-BB-ζ receptors.Fourteen days after transduction a mean (±SD) of 89.6% ±2.3% (n=6) ofGFP⁺ cells also expressed CD3; 66.2%±17.9% of CD3⁺T lymphocytes weretransduced. Among GFP⁺ cells, 21.1%±8.8% (n =6) were CD4⁺, 68.1%±8.1%(n=6) were CD8⁺, 38.1%±16.1% (n=3) were CD28⁺ and 24.2%±11.6% (n=3) wereCD8⁺CD28⁺. These proportions were similar to those obtained with theanti-CD19-c receptors lacking 4-1BB. In this case, 85.4%±11.0% (n=6) ofGFP⁺ cells expressed CD3; 60.8% ±10.1% of CD3⁺ cells were transduced.Among GFP⁺ cells, 18.0%±8.7% (n=6) were CD4⁺, 66.1%±11.7% (n=6) wereCD8⁺, 41.2%±12.2% (n=3) were CD28⁺ and 20.6%±11.3% (n=3) were CD8⁺CD28⁺.In these experiments, median transduction efficiency was 65% (range, 31%to 86%) for anti-CD19-BB-ζ receptors, and 65% (range, 37% to 83%) foranti-CD19-ζ receptors.

The surface expression of the chimeric receptors on GFP⁺ cells wasconfirmed by staining with a goat anti-mouse antibody that reacted withthe scFv portion of anti-CD19. Expression was detectable on most GFP⁺cells and was not detectable on GFP cells and vector-transduced cells.The level of surface expression of anti-CD19-BB-ζ was identical to thatof the receptor lacking 4-1BB. Expression was confirmed by Western blotanalysis; under non-reducing conditions, peripheral blood mononuclearcells transduced with the chimeric receptors expressed them mostly asmonomers, although dimers could be detected.

Signaling Function of Anti-CD19-BB-ζ Chimeric Receptors

To test the functionality of the anti-CD19-BB-ζ chimeric receptor, weused the T-cell line Jurkat and the CD19+ ALL cell line OP-1. Aftertransduction, >95% Jurkat cells were GFP+. Exposure of irradiated OP-1cells to Jurkat cells transduced with anti-CD19-BB-ζ triggeredtranscription of IL-2. Notably, in parallel experiments with Jurkatcells transduced with the anti-CD 19-ζ receptor lacking 4-1BB, the levelof IL-2 transcription was much lower. No IL-2 transcription was detectedin Jurkat cells transduced with the anti-CD19-truncated control receptorlacking CD3ζ.

To identify further changes in molecules associated with T-cellactivation, survival or cytotoxicity induced by anti-CD19-BB-ζreceptors, Jurkat cells were either transduced with these receptors orwith anti-CD19-ζ receptors and then stimulated withparaformadehyde-fixed OP-1 cells. After 12 hours of stimulation, wescreened the cells' gene expression using Affymetrix HG-U133A chips.Genes that were overexpressed by a factor of 2 or higher in cells withanti-CD19-BB-ζ included the member of the TNF family TRAIL, theTNF-receptor member OX40, and IL-3. Overexpression of these moleculesafter stimulation was validated using RT-PCR. In cells bearing theanti-CD 19-ζ receptor, there were no overexpressed genes with a knownfunction associated with T-cells. Therefore, anti-CD19-BB-ζ receptorselicit transcriptional responses that are distinct from those triggeredby receptors lacking 4-1BB.

Expansion of T Cells Expressing Anti-CD19-BB-ζ Receptors in the Presenceof CD19⁺ Cells

To measure the ability of anti-CD19-BB-ζ transduced lymphocytes tosurvive and expand in vitro, we first analyzed primary T cells (obtainedfrom 2 donors), 7 days after transduction. Transduction efficiency withthe 3 receptors was similar: 72% and 67% for anti-CD19-BB-ζ, 63% and 66%for anti-CD19-ζ and 67% and 68% for the truncated anti-CD19 receptor.When cocultured with irradiated OP-1 ALL cells in the absence ofexogenous IL-2, cells transduced with anti-CD19-BB-ζ expanded: afteronly 1 week of culture, GFP⁺ cells recovered were 320% and 413% of inputcells. T cells that expressed the anti-CD19-ζ receptor but lacked 4-1BBsignaling capacity remained viable but showed little expansion (cellrecovery: 111% and 160% of input cells, respectively), whereas thosethat expressed the truncated anti-CD19 receptor underwent apoptosis(<10% of input cells were viable after 1 week). Lymphocytes transducedwith anti-CD19-BB-ζ continued to expand in the presence of irradiatedOP-1 cells. After 3 weeks of culture, they had expanded by more than16-fold, with 98% of the cells at this point being GFP⁺. By contrast,cells transduced with only anti-CD19-ζ survived for less than 2 weeks ofculture.

We performed the next set of experiments with T cells (obtained from 3donors) 14 days after transduction with anti-CD19-BB-ζ, anti-CD19-ζ oranti-CD19-truncated, and expanded with high-dose IL-2 (200 IU/mL).Recovery of lymphocytes of each donor with anti-CD19-BB-ζ receptors wassignificantly higher than that of lymphocytes with anti-CD 19-ζreceptors in all 3 comparisons (P<0.005). When IL-2 was removed,exposure of the transduced cells to irradiated OP-1 cells inducedapoptosis, irrespective of the chimeric receptor expressed. This was incontrast to results with cells 7 days post-transduction, and in accordwith the loss of T cell functionality after prolonged culture in IL-2observed by others. [Brentjens R J, et al. Eradication of systemicB-cell tumors by genetically targeted human T lymphocytes co-stimulatedby CD80 and interleukin-15. Nat Med 9:279-286 (2003); Rossig C. et al.Targeting of G(D2)-positive tumor cells by human T lymphocytesengineered to express chimeric T-cell receptor genes. Int J Cancer94:228-236 (2001)] However, low-dose IL-2 (10 IU/mL) was sufficient tomaintain most lymphocytes transduced with anti-CD19-BB-ζ viable after 2weeks of culture with irradiated OP-1 cells, but did not preventapoptosis of cells transduced with the other receptors. Taken together,these data indicate that 4-1BB-mediated costimulation confers a survivaladvantage on lymphocytes.

Cytotoxicity Triggered by Anti-CD19-BB-ζ Chimeric Receptors

Lymphocytes obtained from two donors and transduced with anti-CD19-BB-ζand anti-CD19-ζ exerted dose-dependent cytotoxicity, as shown by a5-hour LDH release assay using the OP-1 B-lineage ALL cell line as atarget. Transduction efficiencies were 41% and 73% for empty vector, 40%and 67% for anti-CD19-truncated, 43% and 63% for anti-CD19-ζ, and 46%and 72% for anti-CD19-BB-ζ. No differences in cytotoxicities mediated bythe two receptors were detectable with this assay. Although no lysis oftarget cells was apparent at a 1:1 ratio in the 5-hour LDH assay, mostleukemic cells were specifically killed by lymphocytes expressingsignaling chimeric receptors when the cultures were examined at 16 hoursby flow cytometry and inverted microscopy.

To better mimic the application of T-cell therapy, we determined whetherT cells expressing the chimeric receptor would exert significantanti-leukemic activity when present at low E:T ratios in prolongedculture. Lymphocytes from various donors were expanded in vitro for 14days after transduction and were mixed at different ratios with OP-1,RS4;11, or REH B-lineage ALL cells, or with K562 (a CD19-negativemyeloid cell line that lacks HLA antigens) transduced with CD19 or withvector alone. Co-cultures were maintained for 7 days, and viableleukemic cells were counted by flow cytometry. As observed in short termcultures, at a 1:1 ratio, T cells expressing signaling chimericreceptors eliminated virtually all leukemic cells from the cultures. Ata 0.1:1 ratio, however, T cells transduced with anti-CD19-BB-ζ receptorswere markedly more effective than those lacking 4-1BB signaling.Chimeric receptor-transduced T cells had no effect on cells lackingCD19. The presence of 4-1BB in the chimeric receptor did not increasebackground, non-CD19-mediated cytotoxicity, in experiments using CEM-C7,U-937 and K-562. As in other experiments, transduction efficiencies withthe two chimeric receptors were equivalent, and range from 62% to 73%for anti-CD19-ζ and from 60% to 70% for anti-CD19-BB-ζ.

Cells present in the bone marrow microenvironment may decrease T-cellproliferation in a mixed lymphocyte reaction. [Bartholomew A, et al.Mesenchymal stem cells suppress lymphocyte proliferation in vitro andprolong skin graft survival in vivo. Exp Hematol 30:42-48 (2002);Krampera M, et al. Bone marrow mesenchymal stem cells inhibit theresponse of naive and memory antigen-specific T cells to their cognatepeptide. Blood 101:3722-3729 (2003); Le Blanc K, et al. Mesenchymal stemcells inhibit and stimulate mixed lymphocyte cultures and mitogenicresponses independently of the major histocompatibility complex. Scand JImmunol 57:11-20 (2003)] To test whether these cells would also affectT-cell-mediated antileukemic activity, we repeated the experiments withOP-1 in the presence of bone marrow-derived mesenchymal cell layers.[Mihara K, et al. Development and functional characterization of humanbone marrow mesenchymal cells immortalized by enforced expression oftelomerase. Br J Haematol 2003;120:846-849 (2003)] T-cell cytotoxicityunder these conditions was even greater than that observed in cultureswithout mesenchymal cells. Remarkably, T cells transduced withanti-CD19-BB-ζ were markedly cytotoxic even at a ratio of 0.01:1 in thisassay, whereas those transduced with anti-CD19-ζ were not.

Effect of Receptor-Transduced T Cells on Primary Leukemic Cells

We co-cultured primary B-lineage ALL cells with bone marrow-derivedmesenchymal cells, which are essential to preserve their viability invitro. [Nishigaki H, et al. Prevalence and growth characteristics ofmalignant stem cells in B-lineage acute lymphoblastic leukemia. Blood1997;89:3735-3744 (1997); Mihara K, et al. Development and functionalcharacterization of human bone marrow mesenchymal cells immortalized byenforced expression of telomerase. Br J Haematol 120:846-849 (2003)] Wetested the effect of T cells expressing anti-CD19-BB-ζ on primaryleukemic cells obtained from 5 patients at the time of diagnosis; thesepatients included 3 who had B-lineage ALL with 11q23 abnormalities, akaryotype associated with drug resistance. [Pui C H, et al. Childhoodacute lymphoblastic leukemia—Current status and future perspectives.Lancet Oncology 2:597-607 (2001)] Mesenchymal cells supported ALL cellsurvival in vitro: in cultures not exposed to exogenous T cells,recovery of leukemic cells from the 5 patients after 5 days of cultureranged from 100.1% to 180.7% of the input cell number. Leukemic cellsincubated at a 0.1:1 ratio with lymphocytes expressing anti-CD19-BB-ζwere virtually eliminated in all 5 cultures. Remarkable cytotoxicity wasalso seen at a 0.01:1 ratio. Importantly, at this ratio, lymphocytesexpressing anti-CD19-BB-ζ were consistently more cytotoxic than thoseexpressing the anti-CD19-ζ receptor alone (P<0.01 by t test for allcomparisons).

Comparisons Between Chimeric Receptors Containing Signaling Domains of4-1BB and of CD28

We compared responses induced by anti-CD19-BB-ζ to those of anequivalent receptor in which 4-1BB signaling domains were replaced byCD28 signaling domains (FIG. 1). Expression of the latter was similar tothat of anti-CD19-BB-ζ and anti-CD19-ζ receptors: >95% Jurkat cells wereconsistently GFP+after transduction with anti-CD19-28-ζ and most ofthese cells had detectable receptors on the cell surface. In 6experiments with primary lymphocytes, transduced cells ranged from 42%to 84% (median, 72%).

We tested production of IL-2 in Jurkat cells transduced with the threereceptors and stimulated with the CD19+ ALL cell line OP-1. Productionof IL-2 was the highest in cells expressing anti-CD19-BB-ζ (P<0.05).Production of IL-2 was also tested in primary lymphocytes, which weretransduced with the chimeric receptors and then expanded for 5 weekswith pulses of OP-1. The pattern of IL-2 production was similar to thatobserved in Jurkat cells. Cells expressing anti-CD19-BB-ζ producedhigher levels of IL-2 (P<0.01). Chimeric receptors containing theco-stimulatory molecules induced a higher IFN-γ production in primarylymphocytes. IFN-γ levels were the highest with the anti-CD19-28-ζreceptor (P<0.05). Finally, we tested surface expression of TRAILprotein in primary lymphocytes by staining with a specific antibody.Levels of TRAIL were the highest in cells transduced with theanti-CD19-BB-ζ receptor. These results indicate that anti-CD19-BB-ζreceptors are functionally distinct from those lacking co-stimulatorymolecules or containing CD28 instead of 4-1BB.

Next, we compared the cytotoxicity exerted by primary T cells transducedwith anti-CD19-BB-ζ receptors to those exerted by T cells bearingreceptors lacking 4-1BB. For these experiments, we transduced primarylymphocytes from 2 donors with anti-CD19-BB-ζ anti-CD19-28-ζ,anti-CD19-ζ and anti-CD19-truncated, we expanded them for 2-3 weeks withIL-2, and then purified CD8⁺, GFP⁺ cells by fluorescence activated cellsorting. Confirming our previous results with unsorted cells, CD8⁺ cellsexpressing anti-CD19-BB-ζ receptors were significantly more effectivethan those with anti-CD19-ζ receptors, and were as effective as thosewith anti-CD19-BB-ζ Finally, we determined the capacity of the purifiedCD8 cells transduced with the various receptors to expand in thepresence of low dose (10 U/mL) IL-2. Cells transduced withanti-CD19-BB-ζ receptor had a significantly higher cell growth underthese conditions than those bearing the other receptors (P<0.001).

Discussion

Results of this study indicate that anti-CD19-BB-ζ receptors could helpachieve effective T-cell immunotherapy of B-lineage ALL. Lymphocytesexpressing anti-CD19-BB-ζ survived and expanded better than those withequivalent receptors lacking 4-1BB. These lymphocytes also had higheranti-leukemic activity and could kill B-lineage ALL cells from patientsat E:T ratios as low as 0.01:1, suggesting that the infusion ofrelatively low numbers of transduced T cells could have a measurableanti-leukemic effect in patients. Finally, lymphocytes transduced withanti-CD19-BB-ζ were particularly effective in the presence of bonemarrow-derived mesenchymal cells which form the microenvironmentcritical for B-lineage ALL cell growth, further supporting theirpotential for immunotherapy.

Two recently reported studies used anti-CD19 scFv as a component of achimeric receptor for T-cell therapy of B-cell malignancies. Cooper etal. Blood 101:1637-1644 (2003) reported that T-cell clones transducedwith chimeric receptors comprising anti-CD19 scFv and CD3ζ producedapproximately 80% specific lysis of B-cell leukemia and lymphoma celllines at a 1:1 E:T ratio in a 4-hour ⁵¹Cr release assay; at this ratio,percent specific lysis of one primary B-lineage ALL sample tested wasapproximately 30%. Brentjens et al. Nat Med 279-286 (2003) reported thatT-cells bearing anti-CD19 scFv and CD3ζ chimeric receptors could begreatly expanded in the presence of exogenous IL-15 and artificialantigen-presenting cells transduced with CD19 and CD80. The authorsshowed that these T cells significantly improved the survival ofimmunodeficient mice engrafted with the Raji B-cell lymphoma cell line.Their results demonstrated the requirement for co-stimulation inmaximizing T-cell-mediated anti-leukemic activity: only cells expressingthe B7 ligands of CD28 elicited effective T-cell responses. However,B-lineage ALL cells typically do not express B7-1(CD80) and only asubset expresses B7-2 (CD86) molecules. [Cardoso AA, et al. Pre-B acutelymphoblastic leukemia cells may induce T-cell anergy to alloantigen.Blood 88:41-48 (1996)]

4-1BB, a tumor necrosis factor-receptor family member, is aco-stimulatory receptor that can act independently from CD28 to preventactivation-induced death of activated T cells. [Kim Y J, et al. Human4-1BB regulates CD28 co-stimulation to promote Th1 cell responses. Eur JImmunol 28:881-890 (1998); Hurtado J C, et al. Signals through 4-1BB arecostimulatory to previously activated splenic T cells and inhibitactivation-induced cell death. J Immunol 158:2600-2609 (1997);DeBenedette M A, et al. Costimulation of CD28-T lymphocytes by 4-1BBligand. J Immunol 1997;158:551-559 (1997); Bukczynski J, et al.Costimulation of human CD28-T cells by 4-1BB ligand. Eur J Immunol33:446-454 (2003)] In our study, we found that chimeric receptorscontaining 4-1BB can elicit vigorous signals in the absence ofCD28-mediated co-stimulation. Cytotoxicity against CD19⁺ cells mediatedby these receptors was as good as that mediated by CD28-containingreceptors and was clearly superior to that induced by receptors lackingco-stimulatory molecules. It is known that, in contrast to CD28, 4-1BBstimulation results in a much larger proliferation of CD8⁺ cells thanCD4+ cells. [Shuford W W, et al. 4-1BB costimulatory signalspreferentially induce CD8⁺ T cell proliferation and lead to theamplification in vivo of cytotoxic T cell responses. J Exp Med1997;186:47-55 (1997)] We found that T cells expressing theanti-CD19-BB-c receptor produced more IL-2 upon stimulation, and thatCD8⁺ cells expanded in the presence of low-dose IL-2 more vigorouslythan those expressing receptors lacking 4-1BB domains, including thosecontaining CD28. Therefore, the presence of 4-1BB in the chimericreceptors may support more durable T cell responses than those inducedby other receptors.

Experimental evidence indicates that harnessing 4-1BB signaling couldhave useful application in antitumor therapy. Melero et al. Nat Med3:682-685 (1997) found that antibodies to 4-1BB significantly improvedlong-lasting remission and survival rates in mice inoculated with theimmunogenic P815 mastocytoma cell line. Moreover, immunogenic murinetumor cells made to express 4-1BB ligand were readily rejected andinduced long term immunity. [Melero I, et al. Chen L. Amplification oftumor immunity by gene transfer of the co-stimulatory 4-1BB ligand:synergy with the CD28 co-stimulatory pathway. Eur J Immunol 28:1116-1121(1998)] Dramatic results were also observed in vaccination experimentsusing other tumor cell lines expressing 4-1BB ligands. [Ye Z, et al.Gene therapy for cancer using single-chain Fv fragments specific for4-1BB. Nat Med 8:343-348 (2002); Mogi S, et al. Tumour rejection by genetransfer of 4-1BB ligand into a CD80(+) murine squamous cell carcinomaand the requirements of co-stimulatory molecules on tumour and hostcells. Immunology 101:541-547 (2000); Yoshida H, et al. A noveladenovirus expressing human 4-1BB ligand enhances antitumor immunity.Cancer Immunol Immunother 52:97-106 (2003)] Of note, experiments withthe poorly immunogenic Ag104A fibrosarcoma cell line provided someevidence that 4-1BB could be superior to CD28 in eliciting anti-tumorresponses: 80% of mice showed tumor regression with 4-1BB stimulationand 50% of mice with widespread metastasis were cured, [Melero I,Shuford W W, Newby S A, et al. Monoclonal antibodies against the 4-1BBT-cell activation molecule eradicate established tumors. Nat Med3:682-685 (1997)] whereas CD28 costimulation was not effective alone andrequired simultaneous CD2 stimulation. [Li Y, et al. Costimulation byCD48 and B7-1 induces immunity against poorly immunogenic tumors. J ExpMed 1996;183:639-644 (1996)] These data, together with our results,indicate that the addition of 4-1BB to the chimeric receptor shouldsignificantly increase the probability that transduced T-cells willsurvive and continue to proliferate when the receptor is engaged invivo. We think it noteworthy that T cells with chimeric receptorscontaining 4-1BB expressed the highest levels of TRAIL upon stimulation,given the known tumoricidal activity of this molecule. [Schmaltz C, etal. T cells require TRAIL for optimal graft-versus-tumor activity. NatMed 8:1433-1437 (2002)]

Clinical precedents, such as administration of T-cell clones that targetCMV epitopes [Walter E A, et al. Reconstitution of cellular immunityagainst cytomegalovirus in recipients of allogeneic bone marrow bytransfer of T-cell clones from the donor. N Engl J Med. 333:1038-1044(1995)] or EBV-specific antigens, [Rooney C M, et al. Use ofgene-modified virus-specific T lymphocytes to controlEpstein-Barr-virus-related lymphoproliferation. Lancet 345:9-13 (1995)]attest to the clinical feasibility of adoptive T-cell therapy. Transferof chimeric receptor-modified T cells has the added advantage ofpermitting immediate generation of tumor-specific T-cell immunity.Subsequently, therapeutic quantities of antigen-specific T cells can begenerated quite rapidly by exposure to target cells and/or artificialantigen-presenting cells, in the presence of ligands of co-stimulatorymolecules and/or exogenous cytokines such as IL-2, IL-7, and IL-15.[Geiger T L, Jyothi M D. Development and application ofreceptor-modified T lymphocytes for adoptive immunotherapy. Transfus MedRev 15:21-34 (2001); Schumacher T N. T-cell-receptor gene therapy. NatRev Immunol. 2:512-519 (2002); Sadelain M, et al. Targeting tumours withgenetically enhanced T lymphocytes. Nat Rev Cancer 3:35-45 (2003);Brentjens R J, et al. Eradication of systemic B-cell tumors bygenetically targeted human T lymphocytes co-stimulated by CD80 andinterleukin-15. Nat Med 9:279-286 (2003)] A specific risk of thestrategy proposed here relates to the transforming potential of theretrovirus used to transduce chimeric receptors. [Baum C, Dullmann J, LiZ, et al. Side effects of retroviral gene transfer into hematopoieticstem cells. Blood 101:2099-2114 (2003)] We therefore envisage thecoexpression of suicide genes as a safety measure for clinical studies.[Marktel S, et al. Immunologic potential of donor lymphocytes expressinga suicide gene for early immune reconstitution after hematopoieticT-cell-depleted stem cell transplantation. Blood 101:1290-1298 (2003)]This approach would also ensure that the elimination of normal CD19⁺B-lineage cells is temporary and should therefore have limited clinicalconsequences.

In view of the limited effectiveness and the high risk of the currentlyavailable treatment options for chemotherapy-refractory B-lineage ALLand other B cell malignancies, the results of our study providecompelling justification for clinical trials using T cells expressinganti-CD19-BB-ζ receptors. Donor-derived T cells endowed with chimericreceptors could replace infusion of non-specific lymphocytespost-transplant. To reduce the risk of GvHD mediated by endogenousT-cell receptors, it may be beneficial to use T cells with restrictedendogenous specificity, for example, Epstein-Barr-virus-specificcytotoxic T-lymphocyte lines. [Rossig C, et al. Epstein-Barrvirus-specific human T lymphocytes expressing antitumor chimeric T-cellreceptors: potential for improved immunotherapy. Blood. 99:2009-2016(2002)] Therefore, it would be important to test the effects of adding4-1BB to chimeric receptors transduced in these lines. The reinfusion ofautologous T cells collected during clinical remission could also beconsidered in patients with persistent minimal residual disease. In ourexperiments, T cells expressing anti-CD19-BB-ζ receptors completelyeliminated ALL cells at E:T ratios higher than 1:1, and autologous Blymphocytes became undetectable shortly after transduction ofanti-CD19-BB-ζ, suggesting that the potential leukemic cellcontamination in the infused products should be greatly reduced orabrogated by the procedure.

9.2 Example 2

T lymphocytes transduced with anti-CD19 chimeric receptors haveremarkable anti-ALL capacity in vitro and in vivo, suggesting theclinical testing of receptor-modified autologous T cells in patientswith persistent minimal residual disease. However, the use of allogeneicreceptor-modified T lymphocytes after hematopoietic cell transplantation(HCT) might carry the risk of severe graft-versus-host disease (GvHD).In this setting, the use of CD3-negative natural killer (NK) cells isattractive because they should not cause GvHD.

Spontaneous cytotoxicity of NK cells against ALL is weak, if measurableat all. To test whether anti-CD19 chimeric receptors could enhance it,we developed methods to specifically expand human primary NK cells andinduce high levels of receptor expression. Specific NK cell expansionhas been problematic to achieve with established methods which favorCD3+ T cell expansion. Even after T-cell depletion, residual T cellstypically become prominent after stimulation.

We overcame this obstacle by generating a genetically-modified K562myeloid leukemia cell line that expresses membrane-bound interleukin-15(IL-15) and 4-1BB ligand (CD137L) (K562-mb15-137L). The K562-mb15-137cell line was generated by retrovirally transducing K562 cells with achimeric protein construct consisting of human IL-15 mature peptidefused to the signal peptide and transmembrane domain of human CD8alpha,as well as GFP. Transduced cells were single cell-cloned by limitingdilution and a clone with the highest expression of GFP andmembrane-bound (surface) IL-15 was selected. Then, the clone wastransduced with human CD137L.

Peripheral blood mononuclear cells from 8 donors were cultured withK562-mb15-137L in the presence of 10 IU/mL IL-2. After 1 week of culturewith K562-mb15-137L, NK cells expanded by 16.3±5.9 fold, whereas T cellsdid not expand. The stimulatory effect of K562-mb15-137L was much higherthan that of K562 cells transduced with control vectors, K562 expressingmembrane-bound IL-15 or CD137L alone, or K562 expressing wild-type IL-15instead of membrane-bound IL-15.

NK cells expanded with K562-mb15-137L were transduced with a retroviralvector and the anti-CD19-BB-ζ chimeric receptor. In 27 experiments, meantransduction efficiency (±SD) after 7-14 days was 67.5%±16.7%. Seven tofourteen days after transduction, 92.3% (range 84.7%-99.4%) of cellswere CD3- CD56+ NK cells; expression of receptors on the cell surfacewas high. NK cells expressing anti-CD 19-BB-ζ had powerful cytotoxicityagainst NK-resistant B-lineage ALL cells. NK cells transduced withanti-CD19-BB-ζ had consistently higher cytotoxicity than thosetransduced with receptors lacking 4-1BB.

Transduction of NK Cells with Chimeric Receptors

Peripheral blood mononuclear cells were stimulated with theK562-mb15-137L cells prior to their exposure to retroviral vectorscontaining anti-CD 19 receptor constructs and GFP. In 10 experiments,median percent of NK cells was 98.4% (93.7-99.4%) 7-11 days aftertransduction; 77.4% (55.2-90.0%) of these cells were GFP+. We observedhigh levels of surface expression of the anti-CD19 chimeric receptors.

NK activity against the CD19-negative cells K562 and U937 was notaffected by the expression of anti-CD19 receptors. The receptors,however, markedly increased NK activity against CD 19⁺ ALL cells. Thefollowing summarizes results obtained with NK cells from 2 donors. At anE:T ratio of 1:1, NK cells from donor 1 lacked cytotoxicity againstCD19⁺ RS4;11 cells and exerted ˜50% cytoxicity against CD19⁺ 697 cellsafter 24 hours. NK cells from donor 2 had no cytotoxicity against RS4;11or 697 cells. Expression of the anti-CD19-CD3

receptor overcame NK resistance. NK cells from donor 1 became cytotoxicto RS4;11 cells and those from donor 2 become cytotoxic to both RS;11and 697 cells. Moreover, when control cells had some cytotoxicity, thiswas significantly augmented by expression of signaling anti-CD 19receptor.

Subsequently, we found that addition of the co-stimulatory CD28 or 4-1BBto the anti-CD 19 receptor markedly enhanced NK cytotoxicity againstNK-resistant ALL cells (FIG. 2). For example, after 24 hours of cultureat 1:1 E:T ratio, the cytotoxicity mediated by the anti-CD19-BB-ζreceptor against the NK-resistant CD19⁺ ALL cell lines 380, 697,KOPN57bi and OP1 ranged from 86.5% to 99.1%. Therefore, the inclusion ofco-stimulatory molecules enhances not only the cytoxicity of Tlymphocytes but also that of NK cells.

9.3 Example 3 Artificial Antigen Producing Cells (APCs) Pave The Way ForClinical Application By Potent Primary In Vitro Induction Materials AndMethods

Cells

The CD 19 human B-lineage ALL cell lines RS4;11, OP-1, 380, 697, andKOPN57bi; the T-cell line GEM-C7; and the myeloid cell lines K562 andU-937 were available in our laboratory. Cells were maintained inRPMI-1640 (Gibco, Grand Island, N.Y.) supplemented with 10% fetal calfserum (FCS; BioWhittaker, Walkersville, Md.) and antibiotics.

Primary leukemia cells were obtained with appropriate informed consentand Institutional Review Board (M) approval from nine patients withB-lineage ALL; from four of these patients, we also studied (with IRBapproval) cryopreserved peripheral blood samples obtained duringclinical remission. An unequivocal diagnosis of B-lineage ALL wasestablished by morphologic, cytochemical, and immunophenotypic criteria;in each case, more than 95% of the cells were positive for CD19.Peripheral blood was obtained from eight healthy adult donors.Mononuclear cells collected from the samples by centrifugation on aLymphoprep density step (Nycomed, Oslo, Norway) were washed twice inphosphate-buffered saline (PBS) and once in AIM-V medium (Gibco).

Plasmids and Retrovirus Production

The anti-CD 19-ζ, anti-CD19-BB-i and anti-CD19-truncated (control)plasmids are described in Imai, C, et al., Leukemia 18:676-684 (2004).The pMSCV-IRES-GFP, pEQPAM3(-E), and pRDF constructs were obtained fromthe St. Jude Vector Development and Production Shared Resource. Theintracellular domains of human DAP 10, 4-1BB ligand and interleukin-15(IL-15) with long signal peptide were subcloned by polymerase chainreaction (PCR) with a human spleen cDNA library (from Dr. G. Neale, St.Jude Children's Research Hospital) used as a template. An antiCD 19-DAP10 plasmid was constructed by replacing the intracellular domain ofanti-CD 19-ζ with that of DAP 10, using the SOE-PCR (splicing byoverlapping extension by PCR) method. The signal peptide of CD8cc, themature peptide of IL-15 and the transmembrane domain of CDBa wereassembled by SOE-PCR to encode a “membrane-bound” form of IL-15. Theresulting expression cassettes were subcloned into EcoRI and XhoI sitesof MSCV-IRES-GFP.

The RD114-pseudotyped retrovirus was generated as described in Imai, C,et al., Leukemia 18:676-684 (2004). We used calcium phosphate DNAprecipitation to transfect 293T cells with anti-CD19-ζ, anti-CD19-DAP10,anti-CD19-BB-ζ, or anti-CD19-truncated; pEQ-PAM3(-E); and pRDF.Conditioned medium containing retrovirus was harvested at 48 hours and72 hours after transfection, immediately frozen in dry ice, and storedat −80° C. until use.

Development of K562 Derivatives, Expansion of NK Cells and GeneTransduction

K562 cells were transduced with the construct encoding the“membrane-bound” form of IL-15. Cells were cloned by limiting dilution,and a single-cell clone with high expression of GFP and of surface IL-15(“K562-mb 15”) was expanded. This clone was subsequently transduced withhuman 4-1BB ligand and designated as “K562-mb15-41BBL”. K562 cellsexpressing wild-type IL-15 (“K562-wt 15”) or 4-IBBL (“K562-41BBL”) wereproduced by a similar procedure. Peripheral blood mononuclear cells(1.5×106) were incubated in a 24-well tissue culture plate with orwithout 106 K562-derivative stimulator cells in the presence of 10 N/mLhuman IL-2 (National Cancer Institute BRB Preclinical Repository,Rockville, Md.) in RPMI-1640 and 10% FCS.

Mononuclear cells stimulated with K562-mb15-41BBL were transduced withretroviruses, as previously described for T cells [Melero I, et al.,NK1.1 cells express 4-iBB (CDw137) costimulatory molecule and arerequired for tumor immunity elicited by anti-4-1BB monoclonalantibodies. Cell Immunol 190:167-172 (1998)]. Briefly, 14-mLpolypropylene centrifuge tubes (Falcon) were coated with humanfibronectin (100 μg/mL; Sigma, St. Louis, Mo.) or RetroNectin (50 μg/mL;TaKaRa, Otsu, Japan). Prestimulated cells (2×10⁵) were resuspended inthe tubes in 2-3 mL of virus-conditioned medium with polybrene (4 μg/mL;Sigma) and centrifuged at 2400×g for 2 hours (centrifugation was omittedwhen RetroNectin was used). The multiplicity of infection (4 to 6) wasidentical in each experiment comparing the activity of differentchimeric receptors. After centrifugation, cells were left undisturbedfor 24 hours in a humidified incubator at 37° C., 5% CO₂. Thetransduction procedure was repeated on two successive days. After asecond transduction, the cells were re-stimulated with K562-mb 15-4 1BBLin the presence of 10 IU/mL of IL-2. Cells were maintained in RPMI-1640,10% FCS, and 10 IU/mL IL-2.

Detection of Chimeric Receptor Expression and Immunophenotyping

Transduced NK cells were stained with goat anti-mouse (Fab)² polyclonalantibody conjugated with biotin (Jackson Immunoresearch, West Grove,Pa.) followed by streptavidin conjugated to peridinin chlorophyllprotein (PerCP; Becton Dickinson, San Jose, Calif.). For Westernblotting, cells were lysed in RIPA buffer (PBS, 1% Triton-X100, 0.5%sodium deoxycholate, 0.1% SDS) containing 3 μg/mL of pepstatin, 3 μg/mLof leupeptin, 1 mM of PMSF, 2 mM of EDTA, and 5 μg/mL of aprotinin.Centrifuged lysate supernatants were boiled with an equal volume ofloading buffer with or without 0.1 M DTT, and then separated by SDS PAGEon a precast 10-20% gradient acrylamide gel (BioRad, Hercules, Calif.).The proteins were transferred to a PVDF membrane, which was incubatedwith primary mouse anti-human CD3ζ monoclonal antibody (clone 8D3;Pharmingen). Membranes were then washed, incubated with a goatanti-mouse IgG horseradish peroxidase-conjugated second antibody, anddeveloped by using the ECP kit (Pharmacia, Piscataway, N.J.).

The following antibodies were used for immunophenotypic characterizationof expanded and transduced cells: anti-CD3 conjugated to fluoresceinisothiocyanate (FITC), to peridinin chlorophyll protein (PerCP) or toenergy-coupled dye (ECD); anti-CD 10 conjugated to phycoerythrin (PE);anti-CD19 PE; anti-CD22 PE; anti-CD56 FITC, PE or allophycocyanin (AFC);anti-CD 16 CyChrome (antibodies from Becton Dickinson; Pharmingen, SanDiego; or Beckman-Coulter, Miami, Fla.); and anti-CD25 PE (Dako,Carpinteria, Calif.). Surface expression of KIR and NK activationmolecules was determined with specific antibodies conjugated to FIX orPE (from Beckman-Coulter or Becton-Dickinson), as previously described[Brentjens R J, Latouche J B, Santos E, et al. Eradication of systemicB-cell tumors by genetically targeted human T lymphocytes co-stimulatedby CD80 and interleukin-15. Nat Med 9:279-286 (2003)]. Antibody stainingwas detected with a FACScan or a LSR II flow cytomete (BectonDickinson).

Cytotoxicity Assays and Cytokine Production

Target cells (1.5×105) were placed in 96-well U-bottomed tissue cultureplates (Costar, Cambridge, Mass.) and incubated with primary NK cellstransduced with chimeric receptors at various effector:target (E:T)ratios in RPMI-1640 supplemented with 10% FCS; NK cells were culturedwith 1000 U/mL IL-2 for 48 hours before the assay. Cultures wereperformed in the absence of exogenous IL-2. After 4 hours and 24 hours,cells were harvested, labeled with CD10 PE or CD22 PE and CD56 FITC, andassayed by flow cytometry as previously described. The numbers of targetcells recovered from cultures without NK cells were used as a reference.

For cytokine production, primary NK cells (2×10⁵ in 200 μl) expressingchimeric receptors were stimulated with various target cells at a 1:1ratio for 24 hours. The levels of IFN-γ and GM-CSF in cell-free culturesupernatants were determined with a Bio-Plex assay (BioRad).

Statistical Analysis

A test of equality of mean NK expansion with various stimuli wasperformed using analysis of variance for a randomized complete blockdesign with each donor considered a random block. Tukey's honestsignificant difference procedure was used to compute simultaneousconfidence intervals for each pairwise comparison of the differences oftreatment means. Differences in cytotoxicities and cytokine productionamong NK cells bearing different chimeric receptors were analyzed by thepaired Student's t test.

Results

Culture Conditions that Favor the Expansion of Primary NK Cells

To transduce chimeric receptors into primary NK cells, we searched forstimuli that would induce specific NK cell proliferation. In preliminaryexperiments, peripheral blood mononuclear cells of CD3⁺ T lymphocyteswere depleted and the remaining cells were stimulated with IL-2 (1000U/mL) or IL-15 (10 ng/mL). Under these culture conditions there was noexpansion of NK cells, which in fact progressively declined in numbers.With PHA (7 mg/mL) and IL-2 (1000 U/mL) as stimuli, we observed a 2- to5-fold expansion of CD56⁺ CD3⁻ NK cells after 1 week of culture.However, despite the low proportion of contaminating CD3⁺ cells (<2% intwo experiments) at the beginning of the cultures, these cells expandedmore than NK cells (>30-fold expansion), and after 1 week of culturerepresented approximately 35% of the cell population.

NK cells can be stimulated by contact with the human leukemia cell lineK562, which lacks HLA-antigen expression, [Robertson M J, Cameron C,Lazo S, Cochran K J, Voss S D, Ritz J. Costimulation of human naturalkiller cell proliferation: role of accessory cytokines and cellcontact-dependent signals. Nat Immun 15:213-226 (1996)] and geneticallymodified K562 cells have been used to stimulate cytotoxic T lymphocytes[Maus M V, Thomas A K, Leonard D G, et al. Ex vivo expansion ofpolyclonal and antigen-specific cytotoxic T lymphocytes by artificialAPCs expressing ligands for the T-cell receptor, CD28 and 4-1BB. NatBiotechnol 20:143-148 (2002)]. We tested whether the NK-stimulatorycapacity of K562 cells could be increased through enforced expression ofadditional NK-stimulatory molecules, using two molecules that are notexpressed by K562 cells and are known to stimulate NK cells. Onemolecule, the ligand for 4-1BB (4-1BBL), triggers activation signalsafter binding to 4-1BB (CD 137), a signaling molecule expressed on thesurface of NK cells [Melero I, Johnston J V, Shufford W W, Mittler R S,Chen L. NK1.I cells express 4-IBB (CDw137) costimulatory molecule andare required for tumor immunity elicited by anti-4-IBB monoclonalantibodies. Cell Immunol 190:167-172 (1998)]. The other molecule, IL-15,is a cytokine known to promote NK-cell development and the survival ofmature NK cells [Carson WE, Fehniger T A, Haldar S, et al. A potentialrole for interleukin-15 in the regulation of human natural killer cellsurvival J Clin Invest. 99:937-943 (1997); Cooper M A, Bush J E,Fehniger T A, et al. In vivo evidence for a dependence on interleukin 15for survival of natural killer cells. Blood 100:3633-3638 (2002);Fehniger T A, Caligiuri M A. Ontogeny and expansion of human naturalkiller cells: clinical implications. Int Rev Immunol 20:503-534 (2001);Wu J, Lanier L L. Natural killer cells and cancer. Adv Cancer Res90:127-56.:127- 156 (2003)]. Since IL-15 has greater biological activitywhen presented to NK cells bound to IL-15Rα on the cell membrane ofstimulatory cells, rather than in its soluble form, we made a constructcontaining the human IL-15 gene fused to the gene encoding the humanCD8α, transmembrane domain, and used it to transduce K562 cells.Expression of IL-15 on the surface of K562 cells was more than fivetimes higher with the IL-15-CD8α construct than with wild-type IL-15.

To test whether the modified K562 cells expressing both 4-11313L andIL-I5 (K562mb15-41BBL cells) promote NK cell expansion, we culturedperipheral blood mononuclear cells from seven donors in the presence oflow-dose (10 U/mL) IL-2 as well as irradiated K562 cells transduced with4-1BBL and/or IL-15, or with an empty control vector. Expression ofeither 4-1BBL or IL-15 by K562 cells improved the stimulation ofNK-stimulatory capacity of K562 in some cases but not overall, whereassimultaneous expression of both molecules led to a consistent andstriking amplification of NK cells (median recovery of CD56⁺ CD3⁻ cellsat 1 week of culture, 2030% of input cells [range, 1020% -2520%]compared with a median recovery of 250% [range, 150% - 640%] for K562cells lacking 4-1BBL and IL-15; P <0.0001). In 24 experiments with cellsfrom 8 donors, NK-cell expansion after 3 weeks of culture with K562cells expressing both stimulatory molecules ranged from 309-fold to12,409 fold (median, 1089-fold). Neither the modified nor unmodifiedK562 cells caused an expansion of T lymphocytes. Among expanded CD56⁺CD3⁻ NK cells, expression of CD56 was higher than that of unstimulatedcells; expression of CD16 was similar to that seen on unstimulated NKcells (median CD 16+ NK cells in 7 donors: 89% before expansion and 84%after expansion). We also compared the expression of KIR molecules onthe expanded NK cells with that on NK cells before culture, using themonoclonal antibodies CD158a (against KIR 2DL1), CD158b (2DL2), NKBI(3DL1) and NKAT2 (2DL3). The prevalence of NK subsets expressing thesemolecules after expansion resembled that of their counterparts beforeculture, although the level of expression of KIR molecules was higherafter culture. Similar results were obtained for the inhibitory receptorCD94, while expression of the activating receptors NKp30 and NKp44became detectable on most cells after culture. In sum, theimmunophenotype of expanded NK cells reiterated that of activated NKcells, indicating that contact with K562-mb1541BBL cells had stimulatedexpansion of all subsets of NK cells.

Transduction of NK Cells with Chimeric Receptors

Before transducing peripheral blood mononuclear cells with retroviralvectors containing chimeric receptor constructs and GFP, we stimulatedthem with K562-mb15-41BBL cells. In 27 experiments, the medianpercentage of NK cells that were GFP⁺ at 7-11 days after transductionwas 69% (43%-93%). Chimeric receptors were expressed at high levels onthe surface of NK cells and, by Western blotting, were in both monomericand dimeric configurations.

To identify the specific signals required to stimulate NK cells withchimeric receptors, and overcome inhibitory signals mediated by KIRmolecules and other NK inhibitory receptors that bind to HLA class Imolecules, we first compared two types of chimeric receptors containingdifferent signaling domains: CD3ζ, a signal-transducing moleculecontaining three immunoreceptor tyrosine-based activation motifs (ITAMs)and linked to several activating receptors expressed on the surface ofNK cells [Farag SS, Fehniger T A, Ruggeri L, Velardi A, Caligiuri M A.Natural killer cell receptors: new biology and insights into thegraft-versus-leukemia effect. Blood 100:1935-1947 (2002); Moretta L,Moretta A. Unravelling natural killer cell function: triggering andinhibitory human NK receptors. EMBO J 23:255-259 (2004)], and DAP 10, asignal transducing molecule with no ITAMs linked to the activatingreceptor NKG2D and previously shown to trigger NK cytotoxicity [Farag SS, Fehniger T A, Ruggeri L, Velardi A, Caligiuri M A. Natural killercell receptors: new biology and insights into the graft-versus-leukemiaeffect. Blood 100:1935-1947 (2002); Moretta L, Moretta A. Unravellingnatural killer cell function: triggering and inhibitory human NKreceptors. EMBO J 23:255-259 (2004); Billadeau D D, Upshaw J L, Schoon RA, Dick C J, Leibson P J. NKG2D-DAPIO triggers human NK cell-mediatedkilling via a Syk-independent regulatory pathway. Nat ImmuNo. 4:557-564(2003)]. As a control, we used NK cells transduced with a vectorcontaining an antiCDl9 receptor but no signaling molecules or containingGFP alone.

NK cells were challenged with the CD19⁺ leukemic cell lines 380, 697 andRS4;11, all of which express high levels of HLA-class I molecules byantibody staining By genotyping, RS4;11 is Cw4/Cw3, Bw4 and A3; 380 isCw4/Cw4, Bw4; and 697 is Cw3/Cw3. Hence, these cell lines were fullycapable of inhibiting NK cell cytotoxicity via binding to NK inhibitoryreceptors.

Expression of receptors without signaling molecules did not increaseNK-mediated cytotoxicity over that exerted by NK cells transduced withthe vector containing only GFP. By contrast, expression of anti-CD19-ζreceptors markedly enhanced NK cytotoxicity in all experiments,regardless of the intrinsic ability of donor NK cells to kill leukemictargets. For example, 380 cells were highly resistant to NK cells fromdonors 2 and 3, but were killed when these donor cells expressedanti-CD19-ζ receptors. Similar observations were made for RS4; 11 cellsand the NK cells of donor 1 and for 697 cells and NK cells of donor 2.Moreover, the anti-CD 19-ζ receptors led to improved killing of targetcells even when natural cytotoxicity was present. In all experiments,the cytotoxicity triggered by the anti-CD19-ζ receptor was enhanced overthat achieved by replacing CD3ζ with DAP 10 (P<0.001).

4-1BB-Mediated Costimulatory Signals Enhance NK Cytotoxicity

Previous studies have shown that the addition of costimulatory moleculesto chimeric receptors enhances the proliferation and cytotoxicity of Tlymphocytes [Imai C, Mihara K, Andreansky M, Nicholson I C, Pui C H,Campana D. Chimeric receptors with 4-1BB signaling capacity provokepotent cytotoxicity against acute lymphoblastic leukemia. Leukemia18:676-684 (2004)]. Of the two best known costimulatory molecules in Tlymphocytes, CD28 and 4-1BB, only 4-1BB is expressed by NK cells [MeleroI, Johnston J V, Shufford W W, Mittler R S, Chen L. NKLI cells express4-1BB (CDw137) costimulatory molecule and are required for tumorimmunity elicited by anti-4-1BB monoclonal antibodies. Cell Immunol1998;190:167-172 (1998); Lang S, Vujanovic N L, Wollenberg B, WhitesideT L. Absence of B7.1-CD28/CTLA-4mediated co-stimulation in human NKcells. Eur J Immunol 28:780-786 (1998); Goodier M R, Londei M. CD28 isnot directly involved in the response of human CD3CD56+ natural killercells to lipopolysaccharide: a role for T cells. Immunology111:384-390(2004)]. We determined whether the addition of 4-1BB to theanti-CD 19-ζ receptor would enhance NK cytotoxicity. In a 4hour-cytotoxicity assay, cells expressing the 41BB-augmented receptorshowed a markedly better ability to kill CD 19⁺ cells than did cellslacking this modification. The superiority of NK cells bearing theanti-CD19-BB-ζ receptor was also evident in 24-hour assays with NK cellsfrom different donors cultured at a 1:1 ratio with the leukemia celllines 697, KOPN57bi and OP-1.

Next, we determined whether the antileukemic activity of NK cellsexpressing anti-CD19-BB-ζ receptors extended to primary leukemicsamples. In five samples from children with different molecular speciesof ALL, NK cells expressing the 4-1BB receptors exerted strongcytotoxicity that was evident even at low E:T ratios (e.g., <1:1; FIG.7) and uniformly exceeded the activity of NK cells expressing signalingreceptors that lacked 4-1BB. Even when donor NK cells had naturalcytotoxicity against ALL cells and CD3ζ receptor did not improve it,addition of 4-1BB to the receptor significantly enhanced cytotoxicity.Consistent with their increased cytotoxicity, NK cells expressinganti-CD19-BB-ζ mediated more vigorous activation signals. Forty-sixpercent of NK cells bearing this receptor expressed the IL2 receptor achain CD25 after 24 hours of coculture with CD19⁺ ALL cells, comparedwith only 17% of cells expressing the anti-CD19-ζ receptor and <1% forcells expressing receptors that lacked stimulatory capacity. Moreover,anti-CD19-BB-ζ receptors induced a much higher production of IFN-g andGM-CSF upon contact with CD19⁺ cells than did receptors without 41BB.

We asked whether the expression of signaling chimeric receptors wouldaffect spontaneous NK activity against NK-sensitive cell lines notexpressing CD 19. Spontaneous cytotoxicity of NK cells from three donorsagainst the CD19⁻ leukemia cell lines K562, U937 and CEM-C7 was notdiminished by expression of chimeric receptors, with or without 4-1BB.

Anti-CD19 Chimeric Receptors Induce NK Cytotoxicity Against AutologousLeukemic Cells

To determine whether the NK cell expansion and transduction system thatwe developed would be applicable to clinical samples, we studiedperipheral blood samples that had been obtained (and cryopreserved) fromfour patients with childhood B-lineage ALL in clinical remission, 25-56weeks from diagnosis. NK cell expansion occur in all four samples:recovery of after one week of culture with K562-mb15-41BBL cells,recovery of CD56⁺ CD3⁻ NK cells ranged from 1350% to 3680% of the input.

After transduction with chimeric receptors, we tested the cytotoxicityof the NK cells against autologous leukemic lymphoblasts obtained atdiagnosis. Expression of anti-CD19-BB-ζ receptors overcame NK cellresistance of autologous cells; NK cells expressing the receptorsexerted cytotoxicity which was as powerful as that observed withallogeneic targets.

Discussion

In this study, we demonstrated that the resistance of cancer cells to NKcell activity can be overcome by chimeric receptors expressed on primaryNK cells. The stimulatory signals triggered by the receptors uponcontact with target cells predominated over inhibitory signals andinduced powerful cytotoxicity against NK-resistant leukemic cell linesand primary leukemic cells. We found that the type of stimulatory signaldelivered by the chimeric receptor was a key factor in inducingcytotoxicity. Although DAP 10 signaling can elicit NK cytotoxicity,chimeric receptors containing this molecule in our study induced weakerNK cell activity than that generated by CD3ζ-containing receptors,despite identical levels of surface expression. We also found thataddition of the costimulatory molecule 4-1BB to the chimeric receptorsmarkedly augmented cytotoxicity, and that receptors containing both CD3ζand 4-1BB triggered a much more robust NK cell activation and cytokineproduction than did those containing only CD3ζ.

The important contribution of 4-1BB signals agrees with findings thatanti-4- I BB antibodies activate murine NK cells [Pan P Y, et al.,Regulation of dendritic cell function by NK cells: mechanisms underlyingthe synergism in the combination therapy of IL-12 and 4-1BB activation.J Immunol 172:4779-4789 (2004)], and enhance their anti-tumor activity.Leukemic lymphoid cells usually do not express 4-1BB ligand: only 2 of284 diagnostic B-lineage ALL samples studied by gene arrays at ourinstitution expressed 4-I BB ligand transcripts [Yeoh E J, et al.,Classification, subtype discovery, and prediction of outcome inpediatric acute lymphoblastic leukemia by gene expression profiling.Cancer Cell 1:133-143 (2002)]. Hence, 4-1BB signals can be delivered toNK cells only if the molecule is incorporated into the receptor.

Efficient and stable transduction of primary NK cells is notoriouslydifficult, prompting us to devise a new gene transduction method for thepresent study. Most investigators have demonstrated efficient genetransfer only in continuously growing NK cell lines [Roberts M R, etal., Antigen-specific cytolysis by neutrophils and NK cells expressingchimeric immune receptors bearing zeta or gamma signaling domains. JImmunol. 161:375-384 (1998); Nagashima S, et al., Stable transduction ofthe interleukin-2 gene into human natural killer cell lines and theirphenotypic and functional characterization in vitro and in vivo. Blood91:3850-3861(1998)] or reported methods yielding only transient geneexpression [Billadeau D D, et al., NKG2D-DAP 10 triggers human NKcell-mediated killing via a Syk-independent regulatory pathway. NatImmuNo. 4:557-564 (2003); Trompeter H I, et al., Rapid and highlyefficient gene transfer into natural killer cells by nucleofection. JImmunol Methods 274:245-256 (2003); Schroers R, et al., Gene transferinto human T lymphocytes and natural killer cells by Ad5/F35 chimericadenoviral vectors. Exp Hematol 32:536-546(2004)]. We achieved stableexpression of chimeric receptors in primary CD56⁺ CD3⁻ NK cells by usingan RD114-pseudotyped retroviral vector and specifically expandingprimary CD56⁺ CD3⁻ NK cells before they were exposed to the retrovirus,a step that allowed highly efficient gene expression. Although severalcytokines such as IL-2, IL-12 and IL-15 have been reported to stimulateNK cells [Carson W E, et al., A potential role for interleukin-15 in theregulation of human natural killer cell survival J Clin Invest.99:937-943 (1997); Trinchieri G, et al., Response of resting humanperipheral blood natural killer cells to interleukin 2 J Exp Med 1984;160:1147-1169 (1984); Naume B, et al., A comparative study of IL-12(cytotoxic lymphocyte maturation factor)-, IL-2-, and IL-7-inducedeffects on immunomagnetically purified CD56+NK cells. J Immunol148:2429-2436 (1992)], their capacity to induce proliferation of restingCD56⁺ CD3 cells has been poor, unless accessory cells are present in thecultures. Perussia et al. Nat Immun Cell Growth Regul 6:171-188 (1987),found that contact with irradiated B-lymphoblastoid cells induced ashigh as a 25-fold expansion of NK cells after 2 weeks of stimulation,while Miller et al. Blood ;80:2221-2229 (1992) reported an approximate30-fold expansion of NK cells after 18 days of culture with 1000 U/mLIL-2 and monocytes. However, these culture conditions are likely topromote the growth of CD3⁺ T lymphocytes as well as NK cells. Since ourultimate aim is to generate pure preparations for out donor NK cellsdevoid of CD3⁺ T lymphocytes, that can be infused into recipients ofallogeneic hematopoietic stem cell transplants, we searched for methodsthat would maximize NK cell expansion without producing T-cellmitogenicity.

Contact with K562 cells (which lack MHC-class I molecule expression andhence do not trigger KIR-mediated inhibitory signals in NK cells) isknown to augment NK cell proliferation in response to IL-15. We foundthat membrane-bound IL-15 and 4-1BBL, coexpressed by K562 cells, actedsynergistically to augment K562-specific NK stimulatory capacity,resulting in vigorous expansion of peripheral blood CD56⁺ CD3⁻ NK cellswithout concomitant growth of T lymphocytes. After 2-3 weeks of culture,we observed NK cell expansions of up to 10,000-fold, and virtually purepopulations of NK cells could be obtained, even without the need forT-cell depletion in some cases. NK cells expanded in this systemretained the immunophenotypic diversity seen among peripheral bloodsubsets of NK cells, as well as their natural cytotoxicity againstsensitive target cells, even after transduction with different chimericreceptors. Hence, this system should help studies of NK cell biologywhich require specific cell expansion and/or gene transduction, but itshould also be adaptable to clinical applications after generatingK562mb 15-4 1 BBL cells that comply with current good manufacturingpractices for clinical trials. Recently, Harada et al. reported thatexpansions of CD56⁺ CD3⁻ cells (up to 400-fold after 2 weeks) wereapparently superior after contact with another HLA class I-negative cellline, the Wilms tumor cell line HFWT [Harada H, Saijo K, Watanabe S, etal. Selective expansion of human natural killer cells from peripheralblood mononuclear cells by the cell line, HFWT. Jpn J Cancer Res 93:313(2002)]. Future studies should determine whether HFWT cells express 41BBL or whether enforced expression of 4-1BBL together with IL-15 resultsin a greater specific expansion of NK cells than seen with modified K562cells.

In the context of allogeneic hematopoietic stem cell transplantation,infusions of activated donor T cells would carry an unacceptably highrisk of severe GvHD, particularly in recipients of haploidentical ormismatched transplants. By contrast, infusions of pure CD56 CD3 NK cellsshould not impose that risk [Ruggeri L, et al., Effectiveness of donornatural killer cell alloreactivity in mismatched hematopoietictransplants. Science 295 :2097-2100 (2002)]. Most clinical studies ofthe therapeutic effects of NK cells have been performed in an autologoussetting and have yielded only moderately promising results [Farag SS, etal., Natural killer cell receptors: new biology and insights into thegraft-versus-leukemia effect. Blood 100:1935-1947 (2002); Chiorean E G,Miller J S. The biology of natural killer cells and implications fortherapy of human disease. J Hematother Stem Cell Res 10:451-463 (2001)].This is not surprising because NK cell activity is inhibited by surfacereceptors that recognize autologous HLA molecules expressed by bothnormal and neoplastic cells. Allogeneic NK cells may be more effective,but even in an allogeneic setting the capacity of NK cells to killmalignant lymphoid cells is generally modest and often negligible[Caligiuri M A, Velardi A, Scheinberg D A, Borrello I M.Immunotherapeutic approaches for hematologic malignancies. Hematology(Am Soc Hematol Educ Program) 337-353 (2004)]. Leung et al.[ J Immunol172:644-650 (2004)] detected NK cytotoxicity against an ALL cell lineexpressing particularly low levels of inhibitory HLA molecules, butcytotoxicity was much lower than that observed against the NK-celltarget K562: only about 50% of the ALL cells were killed at an effector: target ratio of 40:1. In that study, RS4;11 cells, which express HLA-Calleles that bind the most commonly expressed KIRs, were NK-resistant,whereas these cells, as well as autologous leukemic cells, were highlysensitive to NK cells expressing anti-CD 19 signaling receptors in ourstudy. NK cells expressing signaling chimeric receptors have much morepowerful antileukemic activity than unmodified NK cells, and can killtarget cells irrespective of their HLA profile.An increasedunderstanding of the signals leading to immune cell activation, togetherwith progress in gene cloning and transfer, have made the treatment ofcancer with “adoptively acquired immunity” a realistic goal. Clinicalprecedents, such as administration of T-cell clones that targetcytomegalovirus epitopes [Walter E A, et al., Reconstitution of cellularimmunity against cytomegalovirus in recipients of allogeneic bone marrowby transfer of T-cell clones from the donor. N Engl J Med 1995;333:1038-1044 (1995)] or EBV-specific antigens [Rooney C M, et al., Useof gene-modified virus-specific T lymphocytes to controlEpstein-Barr-virus-related lymphoproliferation. Lancet 345:9-13(1995)],attest to the clinical feasibility of adoptive immune cell therapy.Nonetheless, there are potential limitations that may affect theeffectiveness of cell therapy guided by chimeric receptors. One is thatthe murine scFv portion of the chimeric receptor or the fusion sites ofthe human regions that compose it may trigger a host immune responseleading to elimination of the modified cells [Sadelain M, et al.,Targeting tumours with genetically enhanced T lymphocytes. Nat RevCancer 3:35-45 (2003)]. Although the impact of such an event in aclinical setting remains to be determined, we anticipate that immuneresponses against modified NK cells will be limited in immune-suppressedpatients after hematopoietic stem cell transplantation. Anotherpotential limitation is that adoptively transferred cells may haveinadequate persistence in vivo, although a recent study showed that NKcells obtained from haploidentical donors and activated ex vivo couldexpand in patients when infused after administration of high-dosecyclophosphamide and fludarabine, which caused an increased inendogenous IL-15 [Miller J S, et al., Successful adoptive transfer andin vivo expansion of human haploidentical NK cells in cancer patients.Blood; in press (2005)]. We speculate that such expansions would alsooccur with genetically-modified NK cells, and suggest that furtherstudies to identify signaling molecules that promote NK cellproliferation when incorporated into chimeric receptors are warranted.In patients at a high risk of leukemia or lymphoma relapse, the expectedbenefits of genetically-modified NK cells will outweigh the risk ofinsertional oncogenesis posed by the use of retroviruses for chimericreceptor transduction [Baum C, et al., Side effects of retroviral genetransfer into hematopoietic stem cells. Blood 101:2099-2114 (2003)]. Wealso predict that the coexpression of suicide genes will become a usefulsafety measure in clinical studies [Marktel S, et al., Immunologicpotential of donor lymphocytes expressing a suicide gene for earlyimmune reconstitution after hematopoietic T-cell-depleted stem celltransplantation. Blood 101:1290-1298 (2003)]; this strategy would alsoensure that the elimination of normal CD 19⁺ B-lineage cells is onlytemporary.

Novel therapies that bypass cellular mechanisms of drug resistance areurgently needed for patients with refractory leukemia and lymphoma. NKcell alloreactivity is a powerful new tool for improving the therapeuticpotential of allogeneic hematopoietic stem cell transplantation. Theresults of this study indicate that signaling receptors can enhance theefficacy of NK cell alloreactivity and widen its applicability. Weenvisage initial clinical trials in which donor NK cells, collected byapheresis, are expanded ex vivo as described here, transduced withchimeric receptors and then infused after transplantation in patientswith B-lineage ALL. The target molecule for the chimeric receptors, CD19, was selected because it is one of the most widely expressed surfaceantigens among B-cell malignancies, including ALL, CLL and NHL. In thesemalignancies, CD19 is highly expressed on the surface of virtually allcells but has limited or no expression in normal tissues [Campana D,Behm F G. Immunophenotyping of leukemia. J Immunol Methods 243:59-75(2000)]. However, the NK-cell strategy of immunotherapy we describewould not have to be directed to the CD 19 antigen, but could be appliedto any of the numerous molecules identified as potential targets forchimeric receptor-based cell therapy in cancer patients.

All of the above U.S. patents, U.S. patent application publications,U.S. patent applications, foreign patents, foreign patent applicationsand non-patent publications referred to in this specification, includingbut not limited to U.S. patent application Ser. No. 09/960,264, filedSep. 20, 2001; and U.S. application Ser. No. 10/981,352, filed Nov. 4,2004, are incorporated herein by reference, in their entirety. All ofreferences, patents, patent applications, etc. cited above, areincorporated herein in their entirety.

1. (canceled)
 2. A polynucleotide encoding a chimeric receptorcomprising: (a) an extracellular ligand-binding domain; (b) atransmembrane domain; and (c) a cytoplasmic domain comprising a 4-1BBsignaling domain and a CD3C signaling domain.
 3. The polynucleotide ofclaim 2, wherein said 4-1BB signaling domain is a human 4-1BB signalingdomain.
 4. The polynucleotide of claim 2, wherein said 4-1BB signalingdomain comprises amino acid residues 214-255 of SEQ ID NO:2.
 5. Thepolynucleotide of claim 2, wherein said transmembrane domain comprises atransmembrane domain of CD8α.
 6. The polynucleotide of claim 2, whereinsaid chimeric receptor further comprises a hinge domain.
 7. A vectorcomprising a polynucleotide encoding a chimeric receptor comprising: (a)an extracellular ligand-binding domain; (b) a transmembrane domain; and(c) a cytoplasmic domain comprising a 4-1BB signaling domain and a CD3ζsignaling domain.
 8. The vector of claim 7, wherein said 4-1BB signalingdomain is a human 4-1BB signaling domain.
 9. The vector of claim 7,wherein said 4-1BB signaling domain comprises amino acid residues214-255 of SEQ ID NO:2.
 10. The vector of claim 7, wherein said vectoris an expression vector, and wherein said polynucleotide is operablylinked to at least one regulatory element in the appropriate orientationfor expression.
 11. The vector of claim 10, wherein said vector is aviral vector.
 12. The vector of claim 10, wherein said vector is aretroviral vector.
 13. The vector of claim 7, wherein said chimericreceptor further comprises a hinge domain.
 14. A host cell comprising apolynucleotide encoding a chimeric receptor comprising: (a) anextracellular ligand-binding domain; (b) a transmembrane domain; and (c)a cytoplasmic domain comprising a 4-1BB signaling domain and a CD3ζsignaling domain.
 15. The host cell of claim 14, wherein said 4-1BBsignaling domain is a human 4-1BB signaling domain.
 16. The host cell ofclaim 14, wherein said 4-1BB signaling domain comprises amino acidresidues 214-255 of SEQ ID NO:2.
 17. The host cell of claim 14, whereinsaid polynucleotide further comprises a promoter sequence.
 18. The hostcell of claim 14, wherein said chimeric receptor further comprises ahinge domain.
 19. The host cell of claim 14, wherein said host cell is aT lymphocyte or an NK cell.
 20. The host cell of claim 14, wherein saidhost cell is a T lymphocyte.
 21. The host cell of claim 14, wherein saidhost cell is an NK cell.